GcatWiki - User contributions [en]

Davidson Projects with Updates

2008-07-22T00:05:49Z

JaBarron: /* Building LasI sender '''TESTER''' cell */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== '''TESTING''' Lux Sender & Receiver Cells ==
 
[[Image:setup1.jpg]]
 
[[Image:official.jpg]]
 

== Building LuxR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || ? || ? || ?
|-
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]|| Yes || ? || ? || ?
|} 
[[Image:promoter.jpg]] + [[Image:coding.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 
[[Image:las.jpg]]
 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|S03156|| Yes || Yes || Yes ||
|-
|B0015|| Yes|| Yes|| Yes ||
|-
|R0079|| Yes || Yes || Yes ||
|-
|E0240|| Yes || Yes || Yes ||
|-
|S03954|| N/A || Yes || Yes ||
|-
|S03955|| N/A || Yes || Yes ||
|-
|S03956|| N/A || Yes || Yes ||
|-
|S03966|| N/A || Yes || Yes ||
|-
|K091134|| N/A || Yes || Yes || Ready ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A ([http://partsregistry.org/Part:BBa_S03954 S03954])
[[Image:S03954.jpg]] 
B ([http://partsregistry.org/Part:BBa_S03956 S03956])
[[Image:S03956.jpg]] 
C([http://partsregistry.org/Part:BBa_S03955 S03955])
[[Image:C_1.jpg]]
 
 
Ligation 2: pLacI ([http://partsregistry.org/Part:BBa_R0011 R0011]) + Successful Ligation 1A ([http://partsregistry.org/Part:BBa_S03954 S03954]) 
Done! ('''in pSB1AK3''') 
[[Image:pLacI.jpg]] + [[Image:S03954.jpg]] 
 
Ligation 3: Successful Ligation 2 ([http://partsregistry.org/Part:BBa_S03966 S03966])+ [http://partsregistry.org/Part:BBa_S03956 S03956] 
[[Image:pLacI.jpg]][[Image:S03954.jpg]] + [[Image:S03956.jpg]] 
Done! 
([http://partsregistry.org/Part:BBa_K091134 K091134]) 
[[Image:K091134.jpg]]
 

== Building LasR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_S03954 S03954]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

File:Las.jpg

2008-07-22T00:04:46Z

JaBarron:

Davidson Projects with Updates

2008-07-16T12:39:26Z

JaBarron: /* Building LasR receiver '''TESTER''' cell */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== '''TESTING''' Lux Sender & Receiver Cells ==
 
[[Image:setup1.jpg]]
 
[[Image:official.jpg]]
 

== Building LuxR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || ? || ? || ?
|-
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]|| Yes || ? || ? || ?
|} 
[[Image:promoter.jpg]] + [[Image:coding.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|S03156|| Yes || Yes || Yes ||
|-
|B0015|| Yes|| Yes|| Yes ||
|-
|R0079|| Yes || Yes || Yes ||
|-
|E0240|| Yes || Yes || Yes ||
|-
|S03954|| N/A || Yes || Yes ||
|-
|S03955|| N/A || Yes || Yes ||
|-
|S03956|| N/A || Yes || Yes ||
|-
|S03966|| N/A || Yes || Yes ||
|-
|K091134|| N/A || Yes || Yes || Ready ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A ([http://partsregistry.org/Part:BBa_S03954 S03954])
[[Image:S03954.jpg]] 
B ([http://partsregistry.org/Part:BBa_S03956 S03956])
[[Image:S03956.jpg]] 
C([http://partsregistry.org/Part:BBa_S03955 S03955])
[[Image:C_1.jpg]]
 
 
Ligation 2: pLacI ([http://partsregistry.org/Part:BBa_R0011 R0011]) + Successful Ligation 1A ([http://partsregistry.org/Part:BBa_S03954 S03954]) 
Done! ('''in pSB1AK3''') 
[[Image:pLacI.jpg]] + [[Image:S03954.jpg]] 
 
Ligation 3: Successful Ligation 2 ([http://partsregistry.org/Part:BBa_S03966 S03966])+ [http://partsregistry.org/Part:BBa_S03956 S03956] 
[[Image:pLacI.jpg]][[Image:S03954.jpg]] + [[Image:S03956.jpg]] 
Done! 
([http://partsregistry.org/Part:BBa_K091134 K091134]) 
[[Image:K091134.jpg]]
 

== Building LasR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_S03954 S03954]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

File:K091134.jpg

2008-07-16T12:38:26Z

JaBarron:

Davidson Projects with Updates

2008-07-15T04:36:37Z

JaBarron: /* Building LasR receiver '''TESTER''' cell */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== '''TESTING''' Lux Sender & Receiver Cells ==
 
[[Image:setup1.jpg]]
 
[[Image:official.jpg]]
 

== Building LuxR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || ? || ? || ?
|-
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]|| Yes || ? || ? || ?
|} 
[[Image:promoter.jpg]] + [[Image:coding.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|S03156|| Yes || Yes || Yes ||
|-
|B0015|| Yes|| Yes|| Yes ||
|-
|R0079|| Yes || Yes || Yes ||
|-
|E0240|| Yes || Yes || Yes ||
|-
|S03954|| N/A || Yes || Yes ||
|-
|S03955|| N/A || Yes || Yes ||
|-
|S03956|| N/A || Yes || Yes ||
|-
|S03966|| N/A || Yes || Yes ||
|-
|K091134|| N/A || Yes || Yes || Ready ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A ([http://partsregistry.org/Part:BBa_S03954 S03954])
[[Image:S03954.jpg]] 
B ([http://partsregistry.org/Part:BBa_S03956 S03956])
[[Image:S03956.jpg]] 
C([http://partsregistry.org/Part:BBa_S03955 S03955])
[[Image:C_1.jpg]]
 
 
Ligation 2: pLacI ([http://partsregistry.org/Part:BBa_R0011 R0011]) + Successful Ligation 1A ([http://partsregistry.org/Part:BBa_S03954 S03954]) 
Done! ('''in pSB1AK3''') 
[[Image:pLacI.jpg]] + [[Image:S03954.jpg]] 
 
Ligation 3: Successful Ligation 2 ([http://partsregistry.org/Part:BBa_S03966 S03966])+ [http://partsregistry.org/Part:BBa_S03956 S03956] 
[[Image:pLacI.jpg]][[Image:S03954.jpg]] + [[Image:S03956.jpg]] 
Done! 
 

== Building LasR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_S03954 S03954]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

Davidson Projects with Updates

2008-07-15T04:25:40Z

JaBarron: /* Building LasR receiver '''TESTER''' cell */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== '''TESTING''' Lux Sender & Receiver Cells ==
 
[[Image:setup1.jpg]]
 
[[Image:official.jpg]]
 

== Building LuxR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || ? || ? || ?
|-
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]|| Yes || ? || ? || ?
|} 
[[Image:promoter.jpg]] + [[Image:coding.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|J23100|| Yes || Yes || Pending... ||
|-
|K091118|| Yes|| Yes|| Pending... ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A ([http://partsregistry.org/Part:BBa_S03954 S03954])
[[Image:S03954.jpg]] 
B ([http://partsregistry.org/Part:BBa_S03956 S03956])
[[Image:S03956.jpg]] 
C([http://partsregistry.org/Part:BBa_S03955 S03955])
[[Image:C_1.jpg]]
 
 
Ligation 2: pLacI ([http://partsregistry.org/Part:BBa_R0011 R0011]) + Successful Ligation 1A ([http://partsregistry.org/Part:BBa_S03954 S03954]) 
Done! ('''in pSB1AK3''') 
[[Image:pLacI.jpg]] + [[Image:S03954.jpg]] 
 
Ligation 3: Successful Ligation 2 ([http://partsregistry.org/Part:BBa_S03966 S03966])+ [http://partsregistry.org/Part:BBa_S03956 S03956] 
[[Image:pLacI.jpg]][[Image:S03954.jpg]] + [[Image:S03956.jpg]] 
Done! 
 

== Building LasR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_S03954 S03954]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

Davidson Projects with Updates

2008-07-13T14:47:46Z

JaBarron: /* Building LasR receiver '''TESTER''' cell */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== '''TESTING''' Lux Sender & Receiver Cells ==
 
[[Image:setup1.jpg]]
 
[[Image:official.jpg]]
 

== Building LuxR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || ? || ? || ?
|-
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]|| Yes || ? || ? || ?
|} 
[[Image:promoter.jpg]] + [[Image:coding.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|J23100|| Yes || Yes || Pending... ||
|-
|K091118|| Yes|| Yes|| Pending... ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A ([http://partsregistry.org/Part:BBa_S03954 S03954])
[[Image:S03954.jpg]] 
B ([http://partsregistry.org/Part:BBa_S03956 S03956])
[[Image:S03956.jpg]] 
C([http://partsregistry.org/Part:BBa_S03955 S03955])
[[Image:C_1.jpg]]
 
 
Ligation 2: pLacI ([http://partsregistry.org/Part:BBa_R0011 R0011]) + Successful Ligation 1A ([http://partsregistry.org/Part:BBa_S03954 S03954]) 
Done! ('''in pSB1AK3''') 
[[Image:pLacI.jpg]] + [[Image:S03954.jpg]] 
 
Ligation 3: Successful Ligation 2 ([http://partsregistry.org/Part:BBa_S03966 S03966])+ [http://partsregistry.org/Part:BBa_S03956 S03956] 
[[Image:pLacI.jpg]][[Image:S03954.jpg]] + [[Image:S03956.jpg]] 
Pending...

== Building LasR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_S03954 S03954]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

Davidson Projects with Updates

2008-07-11T20:45:26Z

JaBarron: /* Building LasR receiver '''TESTER''' cell */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== '''TESTING''' Lux Sender & Receiver Cells ==
 
[[Image:setup1.jpg]]
 
[[Image:official.jpg]]
 

== Building LuxR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || ? || ? || ?
|-
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]|| Yes || ? || ? || ?
|} 
[[Image:promoter.jpg]] + [[Image:coding.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|J23100|| Yes || Yes || Pending... ||
|-
|K091118|| Yes|| Yes|| Pending... ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A ([http://partsregistry.org/Part:BBa_S03954 S03954])
[[Image:S03954.jpg]] 
B ([http://partsregistry.org/Part:BBa_S03956 S03956])
[[Image:S03956.jpg]] 
C([http://partsregistry.org/Part:BBa_S03955 S03955])
[[Image:C_1.jpg]]
 
 
Ligation 2: pLacI ([http://partsregistry.org/Part:BBa_R0011 R0011]) + Successful Ligation 1A ([http://partsregistry.org/Part:BBa_S03954 S03954]) 
Done! ('''in pSB1AK3''') 
[[Image:pLacI.jpg]] + [[Image:S03954.jpg]] 
 
Ligation 3: Successful Ligation 2 + [http://partsregistry.org/Part:BBa_S03956 S03956] 
[[Image:pLacI.jpg]][[Image:S03954.jpg]] + [[Image:S03956.jpg]] 
Pending...

== Building LasR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_S03954 S03954]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

File:PLacI.jpg

2008-07-11T20:32:48Z

JaBarron:

Davidson Projects with Updates

2008-07-11T15:55:37Z

JaBarron: /* '''TESTING''' Lux Sender & Receiver Cells */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== '''TESTING''' Lux Sender & Receiver Cells ==
 
[[Image:setup1.jpg]]
 
[[Image:official.jpg]]
 

== Building LuxR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || ? || ? || ?
|-
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]|| Yes || ? || ? || ?
|} 
[[Image:promoter.jpg]] + [[Image:coding.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|J23100|| Yes || Yes || Pending... ||
|-
|K091118|| Yes|| Yes|| Pending... ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A ([http://partsregistry.org/Part:BBa_S03954 S03954])
[[Image:S03954.jpg]] 
B ([http://partsregistry.org/Part:BBa_S03956 S03956])
[[Image:S03956.jpg]] 
C([http://partsregistry.org/Part:BBa_S03955 S03955])
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A ([http://partsregistry.org/Part:BBa_S03954 S03954]) + Successful Ligation 1B ([http://partsregistry.org/Part:BBa_S03956 S03956]) 
Done! 
[http://partsregistry.org/Part:BBa_K091118 K091118][[Image:K091118.jpg]] 
 
Ligation 3: [http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_K091118 K091118] 
[[Image:promoter.jpg]] + [[Image:K091118.jpg]] 
Pending...

== Building LasR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_S03954 S03954]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

File:Official.jpg

2008-07-11T15:54:55Z

JaBarron:

File:Measurement2.jpg

2008-07-11T15:51:03Z

JaBarron:

Davidson Projects with Updates

2008-07-11T14:34:31Z

JaBarron:

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== '''TESTING''' Lux Sender & Receiver Cells ==
 
[[Image:setup1.jpg]]
 
[[Image:measurement1.jpg]]
 

== Building LuxR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || ? || ? || ?
|-
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]|| Yes || ? || ? || ?
|} 
[[Image:promoter.jpg]] + [[Image:coding.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|J23100|| Yes || Yes || Pending... ||
|-
|K091118|| Yes|| Yes|| Pending... ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A ([http://partsregistry.org/Part:BBa_S03954 S03954])
[[Image:S03954.jpg]] 
B ([http://partsregistry.org/Part:BBa_S03956 S03956])
[[Image:S03956.jpg]] 
C([http://partsregistry.org/Part:BBa_S03955 S03955])
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A ([http://partsregistry.org/Part:BBa_S03954 S03954]) + Successful Ligation 1B ([http://partsregistry.org/Part:BBa_S03956 S03956]) 
Done! 
[http://partsregistry.org/Part:BBa_K091118 K091118][[Image:K091118.jpg]] 
 
Ligation 3: [http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_K091118 K091118] 
[[Image:promoter.jpg]] + [[Image:K091118.jpg]] 
Pending...

== Building LasR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_S03954 S03954]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

Davidson Projects with Updates

2008-07-11T14:26:26Z

JaBarron: /* '''Testing''' Lux Sender & Receiver Cells */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== '''Testing''' Lux Sender & Receiver Cells ==
 
[[Image:setup1.jpg]]
 
[[Image:measurement1.jpg]]
 

== Building LuxR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || ? || ? || ?
|-
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]|| Yes || ? || ? || ?
|} 
[[Image:promoter.jpg]] + [[Image:coding.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|J23100|| Yes || Yes || Pending... ||
|-
|K091118|| Yes|| Yes|| Pending... ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A ([http://partsregistry.org/Part:BBa_S03954 S03954])
[[Image:S03954.jpg]] 
B ([http://partsregistry.org/Part:BBa_S03956 S03956])
[[Image:S03956.jpg]] 
C([http://partsregistry.org/Part:BBa_S03955 S03955])
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A ([http://partsregistry.org/Part:BBa_S03954 S03954]) + Successful Ligation 1B ([http://partsregistry.org/Part:BBa_S03956 S03956]) 
Done! 
[http://partsregistry.org/Part:BBa_K091118 K091118][[Image:K091118.jpg]] 
 
Ligation 3: [http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_K091118 K091118] 
[[Image:promoter.jpg]] + [[Image:K091118.jpg]] 
Pending...

== Building LasR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_S03954 S03954]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

Davidson Projects with Updates

2008-07-11T14:25:48Z

JaBarron: /* '''Testing''' Lux Sender & Receiver Cells */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== '''Testing''' Lux Sender & Receiver Cells ==
 
[[Image:setup1.jpg]]
 
[[Image:measurement1.jpg]]

== Building LuxR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || ? || ? || ?
|-
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]|| Yes || ? || ? || ?
|} 
[[Image:promoter.jpg]] + [[Image:coding.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|J23100|| Yes || Yes || Pending... ||
|-
|K091118|| Yes|| Yes|| Pending... ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A ([http://partsregistry.org/Part:BBa_S03954 S03954])
[[Image:S03954.jpg]] 
B ([http://partsregistry.org/Part:BBa_S03956 S03956])
[[Image:S03956.jpg]] 
C([http://partsregistry.org/Part:BBa_S03955 S03955])
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A ([http://partsregistry.org/Part:BBa_S03954 S03954]) + Successful Ligation 1B ([http://partsregistry.org/Part:BBa_S03956 S03956]) 
Done! 
[http://partsregistry.org/Part:BBa_K091118 K091118][[Image:K091118.jpg]] 
 
Ligation 3: [http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_K091118 K091118] 
[[Image:promoter.jpg]] + [[Image:K091118.jpg]] 
Pending...

== Building LasR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_S03954 S03954]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

File:Measurement1.jpg

2008-07-11T14:25:22Z

JaBarron:

File:Setup1.jpg

2008-07-11T14:24:28Z

JaBarron:

Davidson Projects with Updates

2008-07-11T14:21:51Z

JaBarron: /* '''Testing''' Lux Sender & Receiver Cells */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== '''Testing''' Lux Sender & Receiver Cells ==
 
 

== Building LuxR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || ? || ? || ?
|-
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]|| Yes || ? || ? || ?
|} 
[[Image:promoter.jpg]] + [[Image:coding.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|J23100|| Yes || Yes || Pending... ||
|-
|K091118|| Yes|| Yes|| Pending... ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A ([http://partsregistry.org/Part:BBa_S03954 S03954])
[[Image:S03954.jpg]] 
B ([http://partsregistry.org/Part:BBa_S03956 S03956])
[[Image:S03956.jpg]] 
C([http://partsregistry.org/Part:BBa_S03955 S03955])
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A ([http://partsregistry.org/Part:BBa_S03954 S03954]) + Successful Ligation 1B ([http://partsregistry.org/Part:BBa_S03956 S03956]) 
Done! 
[http://partsregistry.org/Part:BBa_K091118 K091118][[Image:K091118.jpg]] 
 
Ligation 3: [http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_K091118 K091118] 
[[Image:promoter.jpg]] + [[Image:K091118.jpg]] 
Pending...

== Building LasR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_S03954 S03954]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

Davidson Projects with Updates

2008-07-11T14:14:27Z

JaBarron: /* '''Testing''' Lux Sender & Receiver Cells */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== '''Testing''' Lux Sender & Receiver Cells ==
 
[[Image:setup.jpg]]
 
[[Image:measurement.jpg]]

== Building LuxR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || ? || ? || ?
|-
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]|| Yes || ? || ? || ?
|} 
[[Image:promoter.jpg]] + [[Image:coding.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|J23100|| Yes || Yes || Pending... ||
|-
|K091118|| Yes|| Yes|| Pending... ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A ([http://partsregistry.org/Part:BBa_S03954 S03954])
[[Image:S03954.jpg]] 
B ([http://partsregistry.org/Part:BBa_S03956 S03956])
[[Image:S03956.jpg]] 
C([http://partsregistry.org/Part:BBa_S03955 S03955])
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A ([http://partsregistry.org/Part:BBa_S03954 S03954]) + Successful Ligation 1B ([http://partsregistry.org/Part:BBa_S03956 S03956]) 
Done! 
[http://partsregistry.org/Part:BBa_K091118 K091118][[Image:K091118.jpg]] 
 
Ligation 3: [http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_K091118 K091118] 
[[Image:promoter.jpg]] + [[Image:K091118.jpg]] 
Pending...

== Building LasR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_S03954 S03954]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

File:Measurement.jpg

2008-07-11T14:12:31Z

JaBarron:

File:Setup.jpg

2008-07-11T14:11:17Z

JaBarron:

Davidson Projects with Updates

2008-07-11T13:50:33Z

JaBarron: /* Testing Lux Sender & Receiver Cells */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== '''Testing''' Lux Sender & Receiver Cells ==
 

== Building LuxR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || ? || ? || ?
|-
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]|| Yes || ? || ? || ?
|} 
[[Image:promoter.jpg]] + [[Image:coding.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|J23100|| Yes || Yes || Pending... ||
|-
|K091118|| Yes|| Yes|| Pending... ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A ([http://partsregistry.org/Part:BBa_S03954 S03954])
[[Image:S03954.jpg]] 
B ([http://partsregistry.org/Part:BBa_S03956 S03956])
[[Image:S03956.jpg]] 
C([http://partsregistry.org/Part:BBa_S03955 S03955])
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A ([http://partsregistry.org/Part:BBa_S03954 S03954]) + Successful Ligation 1B ([http://partsregistry.org/Part:BBa_S03956 S03956]) 
Done! 
[http://partsregistry.org/Part:BBa_K091118 K091118][[Image:K091118.jpg]] 
 
Ligation 3: [http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_K091118 K091118] 
[[Image:promoter.jpg]] + [[Image:K091118.jpg]] 
Pending...

== Building LasR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_S03954 S03954]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

Davidson Projects with Updates

2008-07-11T13:50:05Z

JaBarron:

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== Testing Lux Sender & Receiver Cells ==
 

== Building LuxR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || ? || ? || ?
|-
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]|| Yes || ? || ? || ?
|} 
[[Image:promoter.jpg]] + [[Image:coding.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|J23100|| Yes || Yes || Pending... ||
|-
|K091118|| Yes|| Yes|| Pending... ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A ([http://partsregistry.org/Part:BBa_S03954 S03954])
[[Image:S03954.jpg]] 
B ([http://partsregistry.org/Part:BBa_S03956 S03956])
[[Image:S03956.jpg]] 
C([http://partsregistry.org/Part:BBa_S03955 S03955])
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A ([http://partsregistry.org/Part:BBa_S03954 S03954]) + Successful Ligation 1B ([http://partsregistry.org/Part:BBa_S03956 S03956]) 
Done! 
[http://partsregistry.org/Part:BBa_K091118 K091118][[Image:K091118.jpg]] 
 
Ligation 3: [http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_K091118 K091118] 
[[Image:promoter.jpg]] + [[Image:K091118.jpg]] 
Pending...

== Building LasR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_S03954 S03954]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

Davidson Projects with Updates

2008-07-10T15:34:37Z

JaBarron: /* Building LasR receiver '''TESTER''' cell */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== Building LuxR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || ? || ? || ?
|-
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]|| Yes || ? || ? || ?
|} 
[[Image:promoter.jpg]] + [[Image:coding.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|J23100|| Yes || Yes || Pending... ||
|-
|K091118|| Yes|| Yes|| Pending... ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A ([http://partsregistry.org/Part:BBa_S03954 S03954])
[[Image:S03954.jpg]] 
B ([http://partsregistry.org/Part:BBa_S03956 S03956])
[[Image:S03956.jpg]] 
C([http://partsregistry.org/Part:BBa_S03955 S03955])
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A ([http://partsregistry.org/Part:BBa_S03954 S03954]) + Successful Ligation 1B ([http://partsregistry.org/Part:BBa_S03956 S03956]) 
Done! 
[http://partsregistry.org/Part:BBa_K091118 K091118][[Image:K091118.jpg]] 
 
Ligation 3: [http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_K091118 K091118] 
[[Image:promoter.jpg]] + [[Image:K091118.jpg]] 
Pending...

== Building LasR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_S03954 S03954]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

Davidson Projects with Updates

2008-07-10T15:23:55Z

JaBarron: /* Building LasR receiver '''TESTER''' cell */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== Building LuxR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || ? || ? || ?
|-
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]|| Yes || ? || ? || ?
|} 
[[Image:promoter.jpg]] + [[Image:coding.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|J23100|| Yes || Yes || Pending... ||
|-
|K091118|| Yes|| Yes|| Pending... ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A
[[Image:S03954.jpg]] 
B
[[Image:S03956.jpg]] 
C
[[Image:C_1.jpg]]
 
 
Ligation 2 ([http://partsregistry.org/Part:BBa_K091118 K091118]): Successful Ligation 1A + Successful Ligation 1B 
Done! 
[[Image:K091118.jpg]] 
 
Ligation 3: [http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_K091118 K091118] 
[[Image:promoter.jpg]] + [[Image:K091118.jpg]] 
Pending...

== Building LasR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_S03954 S03954]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

Davidson Projects with Updates

2008-07-10T14:04:27Z

JaBarron: /* Building LasR constitutive protein */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== Building LuxR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || ? || ? || ?
|-
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]|| Yes || ? || ? || ?
|} 
[[Image:promoter.jpg]] + [[Image:coding.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|S03156|| Yes || Yes || Yes ||
|-
|B0015|| Yes|| Yes|| Yes ||
|-
|R0079|| Yes|| Yes || Yes ||
|-
|E0240|| Yes|| Yes || Yes ||
|-
|R0011|| Yes|| Yes|| Pending ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A
[[Image:S03954.jpg]] 
B
[[Image:S03956.jpg]] 
C
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A + Successful Ligation 1B 
Done! 
[[Image:K091118.jpg]] 
 
Ligation 3: R0011 + Ligation 2 

== Building LasR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/Part:BBa_S03954 S03954]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

Davidson Projects with Updates

2008-07-10T13:59:14Z

JaBarron: /* Building LuxR constitutive protein */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== Building LuxR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || ? || ? || ?
|-
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]|| Yes || ? || ? || ?
|} 
[[Image:promoter.jpg]] + [[Image:coding.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|S03156|| Yes || Yes || Yes ||
|-
|B0015|| Yes|| Yes|| Yes ||
|-
|R0079|| Yes|| Yes || Yes ||
|-
|E0240|| Yes|| Yes || Yes ||
|-
|R0011|| Yes|| Yes|| Pending ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A
[[Image:S03954.jpg]] 
B
[[Image:S03956.jpg]] 
C
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A + Successful Ligation 1B 
Done! 
[[Image:K091118.jpg]] 
 
Ligation 3: R0011 + Ligation 2 

== Building LasR constitutive protein ==
Promoter [http://partsregistry.org/Part:BBa_J23100 J23100]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

File:Coding.jpg

2008-07-10T13:58:34Z

JaBarron:

Davidson Projects with Updates

2008-07-10T13:57:58Z

JaBarron: /* Building LuxR constitutive protein */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== Building LuxR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || ? || ? || ?
|-
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]|| Yes || ? || ? || ?
|} 
[[Image:promoter.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|S03156|| Yes || Yes || Yes ||
|-
|B0015|| Yes|| Yes|| Yes ||
|-
|R0079|| Yes|| Yes || Yes ||
|-
|E0240|| Yes|| Yes || Yes ||
|-
|R0011|| Yes|| Yes|| Pending ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A
[[Image:S03954.jpg]] 
B
[[Image:S03956.jpg]] 
C
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A + Successful Ligation 1B 
Done! 
[[Image:K091118.jpg]] 
 
Ligation 3: R0011 + Ligation 2 

== Building LasR constitutive protein ==
Promoter [http://partsregistry.org/Part:BBa_J23100 J23100]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

File:Promoter.jpg

2008-07-10T13:57:24Z

JaBarron:

Davidson Projects with Updates

2008-07-10T13:51:07Z

JaBarron: /* Building LuxR constitutive protein */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== Building LuxR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || ? || ? || ?
|-
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]|| Yes || ? || ? || ?
|} 
[[Image:Const_LuxR.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|S03156|| Yes || Yes || Yes ||
|-
|B0015|| Yes|| Yes|| Yes ||
|-
|R0079|| Yes|| Yes || Yes ||
|-
|E0240|| Yes|| Yes || Yes ||
|-
|R0011|| Yes|| Yes|| Pending ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A
[[Image:S03954.jpg]] 
B
[[Image:S03956.jpg]] 
C
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A + Successful Ligation 1B 
Done! 
[[Image:K091118.jpg]] 
 
Ligation 3: R0011 + Ligation 2 

== Building LasR constitutive protein ==
Promoter [http://partsregistry.org/Part:BBa_J23100 J23100]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

Davidson Projects with Updates

2008-07-10T13:49:28Z

JaBarron: /* Building LuxR constitutive protein */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== Building LuxR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100] + [http://partsregistry.org/wiki/index.php?title=Part:BBa_J37033 J37033]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03954 Fix This]|| ? || ? || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 Fix This]|| ? || ? || ? || ?
|} 
[[Image:Const_LuxR.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|S03156|| Yes || Yes || Yes ||
|-
|B0015|| Yes|| Yes|| Yes ||
|-
|R0079|| Yes|| Yes || Yes ||
|-
|E0240|| Yes|| Yes || Yes ||
|-
|R0011|| Yes|| Yes|| Pending ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A
[[Image:S03954.jpg]] 
B
[[Image:S03956.jpg]] 
C
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A + Successful Ligation 1B 
Done! 
[[Image:K091118.jpg]] 
 
Ligation 3: R0011 + Ligation 2 

== Building LasR constitutive protein ==
Promoter [http://partsregistry.org/Part:BBa_J23100 J23100]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

Davidson Projects with Updates

2008-07-10T13:48:27Z

JaBarron: /* Building LuxR constitutive protein */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== Building LuxR constitutive protein ==
[http://partsregistry.org/Part:BBa_J23100 J23100]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03954 Fix This]|| ? || ? || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 Fix This]|| ? || ? || ? || ?
|} 
[[Image:Const_LuxR.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|S03156|| Yes || Yes || Yes ||
|-
|B0015|| Yes|| Yes|| Yes ||
|-
|R0079|| Yes|| Yes || Yes ||
|-
|E0240|| Yes|| Yes || Yes ||
|-
|R0011|| Yes|| Yes|| Pending ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A
[[Image:S03954.jpg]] 
B
[[Image:S03956.jpg]] 
C
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A + Successful Ligation 1B 
Done! 
[[Image:K091118.jpg]] 
 
Ligation 3: R0011 + Ligation 2 

== Building LasR constitutive protein ==
Promoter [http://partsregistry.org/Part:BBa_J23100 J23100]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

Davidson Projects with Updates

2008-07-10T13:37:06Z

JaBarron: /* Building LuxR constitutive protein */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== Building LuxR constitutive protein ==
Promoter (pBad?) + [http://partsregistry.org/Part:BBa_ Fix this]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03954 Fix This]|| ? || ? || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 Fix This]|| ? || ? || ? || ?
|} 
[[Image:Const_LuxR.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|S03156|| Yes || Yes || Yes ||
|-
|B0015|| Yes|| Yes|| Yes ||
|-
|R0079|| Yes|| Yes || Yes ||
|-
|E0240|| Yes|| Yes || Yes ||
|-
|R0011|| Yes|| Yes|| Pending ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A
[[Image:S03954.jpg]] 
B
[[Image:S03956.jpg]] 
C
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A + Successful Ligation 1B 
Done! 
[[Image:K091118.jpg]] 
 
Ligation 3: R0011 + Ligation 2 

== Building LasR constitutive protein ==
Promoter [http://partsregistry.org/Part:BBa_J23100 J23100]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

Davidson Projects with Updates

2008-07-10T13:14:28Z

JaBarron: /* Building LasR constitutive protein */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== Building LuxR constitutive protein ==
Promoter (pBad?) + [http://partsregistry.org/Part:BBa_ Fix this]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03954 Fix This]|| ? || ? || ? || ?
|-
|} 
[[Image:Const_LuxR.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|S03156|| Yes || Yes || Yes ||
|-
|B0015|| Yes|| Yes|| Yes ||
|-
|R0079|| Yes|| Yes || Yes ||
|-
|E0240|| Yes|| Yes || Yes ||
|-
|R0011|| Yes|| Yes|| Pending ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A
[[Image:S03954.jpg]] 
B
[[Image:S03956.jpg]] 
C
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A + Successful Ligation 1B 
Done! 
[[Image:K091118.jpg]] 
 
Ligation 3: R0011 + Ligation 2 

== Building LasR constitutive protein ==
Promoter [http://partsregistry.org/Part:BBa_J23100 J23100]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]|| Yes || Yes || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

Davidson Projects with Updates

2008-07-10T13:11:31Z

JaBarron: /* Building LuxR receiver '''TESTER''' cell */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== Building LuxR constitutive protein ==
Promoter (pBad?) + [http://partsregistry.org/Part:BBa_ Fix this]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03954 Fix This]|| ? || ? || ? || ?
|-
|} 
[[Image:Const_LuxR.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|S03156|| Yes || Yes || Yes ||
|-
|B0015|| Yes|| Yes|| Yes ||
|-
|R0079|| Yes|| Yes || Yes ||
|-
|E0240|| Yes|| Yes || Yes ||
|-
|R0011|| Yes|| Yes|| Pending ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A
[[Image:S03954.jpg]] 
B
[[Image:S03956.jpg]] 
C
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A + Successful Ligation 1B 
Done! 
[[Image:K091118.jpg]] 
 
Ligation 3: R0011 + Ligation 2 

== Building LasR constitutive protein ==
Promoter [http://partsregistry.org/Part:BBa_J23100 J23100]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]||? || ? || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

Davidson Projects with Updates

2008-07-10T13:11:01Z

JaBarron: /* Building LuxR receiver '''TESTER''' cell */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
 

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== Building LuxR constitutive protein ==
Promoter (pBad?) + [http://partsregistry.org/Part:BBa_ Fix this]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03954 Fix This]|| ? || ? || ? || ?
|-
|} 
[[Image:Const_LuxR.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|S03156|| Yes || Yes || Yes ||
|-
|B0015|| Yes|| Yes|| Yes ||
|-
|R0079|| Yes|| Yes || Yes ||
|-
|E0240|| Yes|| Yes || Yes ||
|-
|R0011|| Yes|| Yes|| Pending ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A
[[Image:S03954.jpg]] 
B
[[Image:S03956.jpg]] 
C
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A + Successful Ligation 1B 
Done! 
[[Image:K091118.jpg]] 
 
Ligation 3: R0011 + Ligation 2 

== Building LasR constitutive protein ==
Promoter [http://partsregistry.org/Part:BBa_J23100 J23100]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]||? || ? || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

Davidson Projects with Updates

2008-07-10T13:10:41Z

JaBarron: /* Building LuxI sender '''TESTER''' cell */

== Building LuxI sender '''TESTER''' cell ==
 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
DONE! (ready to test) 

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== Building LuxR constitutive protein ==
Promoter (pBad?) + [http://partsregistry.org/Part:BBa_ Fix this]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03954 Fix This]|| ? || ? || ? || ?
|-
|} 
[[Image:Const_LuxR.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|S03156|| Yes || Yes || Yes ||
|-
|B0015|| Yes|| Yes|| Yes ||
|-
|R0079|| Yes|| Yes || Yes ||
|-
|E0240|| Yes|| Yes || Yes ||
|-
|R0011|| Yes|| Yes|| Pending ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A
[[Image:S03954.jpg]] 
B
[[Image:S03956.jpg]] 
C
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A + Successful Ligation 1B 
Done! 
[[Image:K091118.jpg]] 
 
Ligation 3: R0011 + Ligation 2 

== Building LasR constitutive protein ==
Promoter [http://partsregistry.org/Part:BBa_J23100 J23100]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]||? || ? || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|} 
'''Testing Lac promoters and proteins''' 
''Construct One'' 
[[Image:Construct12.jpg]] 
''Construct Two'' 
[[Image:Construct2.jpg]] 
-We will test by ligating each of these constructs together with the 3 different lac promoters and the 4 different Lac proteins. We will measure the output of GFP to characterize the strength of each promoter and protein. We will have 12 constructs all together.

Needed From Davidson

2008-07-09T16:28:42Z

JaBarron:

'''Parts Needed ''From'' Davidson'''

'''LuxR expression cassette''' (promoter+RBS+LuxR+TT) '''James Barron''' 
(ligations pending...)

'''LuxI gene''' (RBS+LuxI)
(This is listed in the registry with a promoter Part#S03608[plated and tested SUCCESSFULLY]) 
'''Pallavi Penumetcha'''

'''lasR expression cassette''' (promoter+RBS+LasR+TT) '''James Barron''' 
(complete)

'''LacI gene double mutant''' '''Can we get this gene from Pallavi?'''

'''low copy amp vector''' (I150042 in I51020) '''Samantha''' (hopefully)

'''medium copy amp vector''' (I50032 in I51020) '''Samantha'''

l'''ow copy kan vector''' (not built yet) '''Samantha'''

'''medium copy kan vector''' (not built yet) '''Samantha'''

Davidson Projects with Updates

2008-07-09T14:33:17Z

JaBarron: /* Building LasR constitutive protein */

== Building LuxI sender '''TESTER''' cell ==
DONE! (ready to test) 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
DONE! (ready to test) 

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== Building LuxR constitutive protein ==
Promoter (pBad?) + [http://partsregistry.org/Part:BBa_ Fix this]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03954 Fix This]|| ? || ? || ? || ?
|-
|} 
[[Image:Const_LuxR.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|S03156|| Yes || Yes || Yes ||
|-
|B0015|| Yes|| Yes|| Yes ||
|-
|R0079|| Yes|| Yes || Yes ||
|-
|E0240|| Yes|| Yes || Yes ||
|-
|R0011|| Yes|| Yes|| Pending ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A
[[Image:S03954.jpg]] 
B
[[Image:S03956.jpg]] 
C
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A + Successful Ligation 1B 
Done! 
[[Image:K091118.jpg]] 
 
Ligation 3: R0011 + Ligation 2 

== Building LasR constitutive protein ==
Promoter [http://partsregistry.org/Part:BBa_J23100 J23100]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]||? || ? || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] + [[Image:S03954.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|}

Davidson Projects with Updates

2008-07-09T14:32:22Z

JaBarron: /* Building LasR constitutive protein */

== Building LuxI sender '''TESTER''' cell ==
DONE! (ready to test) 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
DONE! (ready to test) 

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== Building LuxR constitutive protein ==
Promoter (pBad?) + [http://partsregistry.org/Part:BBa_ Fix this]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03954 Fix This]|| ? || ? || ? || ?
|-
|} 
[[Image:Const_LuxR.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|S03156|| Yes || Yes || Yes ||
|-
|B0015|| Yes|| Yes|| Yes ||
|-
|R0079|| Yes|| Yes || Yes ||
|-
|E0240|| Yes|| Yes || Yes ||
|-
|R0011|| Yes|| Yes|| Pending ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A
[[Image:S03954.jpg]] 
B
[[Image:S03956.jpg]] 
C
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A + Successful Ligation 1B 
Done! 
[[Image:K091118.jpg]] 
 
Ligation 3: R0011 + Ligation 2 

== Building LasR constitutive protein ==
Promoter [http://partsregistry.org/Part:BBa_J23100 J23100]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]||? || ? || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:J23100.jpg]] +

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|}

File:J23100.jpg

2008-07-09T14:31:44Z

JaBarron:

Needed From Davidson

2008-07-08T11:48:36Z

JaBarron:

'''Parts Needed ''From'' Davidson'''

'''LuxR expression cassette''' (promoter+RBS+LuxR+TT) '''James Barron''' 
(Part #'''I13018''' [07] Colonies Growing)

'''LuxI gene''' (RBS+LuxI)
(This is listed in the registry with a promoter Part#S03608[plated and tested SUCCESSFULLY]) 
'''Pallavi Penumetcha'''

'''lasR expression cassette''' (promoter+RBS+LasR+TT) '''James Barron''' 
(In progress...)

'''low copy amp vector''' (I150042 in I51020) '''Samantha''' (hopefully)

'''medium copy amp vector''' (I50032 in I51020) '''Samantha'''

l'''ow copy kan vector''' (not built yet) '''Samantha'''

'''medium copy kan vector''' (not built yet) '''Samantha'''

Davidson Projects with Updates

2008-07-07T22:19:29Z

JaBarron: /* Building LasR constitutive protein */

== Building LuxI sender '''TESTER''' cell ==
DONE! (ready to test) 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
DONE! (ready to test) 

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== Building LuxR constitutive protein ==
Promoter (pBad?) + [http://partsregistry.org/Part:BBa_ Fix this]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03954 Fix This]|| ? || ? || ? || ?
|-
|} 
[[Image:Const_LuxR.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|S03156|| Yes || Yes || Yes ||
|-
|B0015|| Yes|| Yes|| Yes ||
|-
|R0079|| Yes|| Yes || Yes ||
|-
|E0240|| Yes|| Yes || Yes ||
|-
|R0011|| Yes|| Yes|| Pending ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A
[[Image:S03954.jpg]] 
B
[[Image:S03956.jpg]] 
C
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A + Successful Ligation 1B 
Done! 
[[Image:K091118.jpg]] 
 
Ligation 3: R0011 + Ligation 2 

== Building LasR constitutive protein ==
Promoter [http://partsregistry.org/Part:BBa_J23100 J23100]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]||? || ? || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|}

Davidson Projects with Updates

2008-07-07T22:13:52Z

JaBarron: /* Building LasR constitutive protein */

== Building LuxI sender '''TESTER''' cell ==
DONE! (ready to test) 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
DONE! (ready to test) 

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== Building LuxR constitutive protein ==
Promoter (pBad?) + [http://partsregistry.org/Part:BBa_ Fix this]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03954 Fix This]|| ? || ? || ? || ?
|-
|} 
[[Image:Const_LuxR.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|S03156|| Yes || Yes || Yes ||
|-
|B0015|| Yes|| Yes|| Yes ||
|-
|R0079|| Yes|| Yes || Yes ||
|-
|E0240|| Yes|| Yes || Yes ||
|-
|R0011|| Yes|| Yes|| Pending ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A
[[Image:S03954.jpg]] 
B
[[Image:S03956.jpg]] 
C
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A + Successful Ligation 1B 
Done! 
[[Image:K091118.jpg]] 
 
Ligation 3: R0011 + Ligation 2 

== Building LasR constitutive protein ==
Promoter [http://partsregistry.org/Part:BBa_J23100 J23100]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_J23100 J23100]| ? || ? || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:Const_LasR.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|}

Needed From Davidson

2008-07-07T19:07:20Z

JaBarron:

'''Parts Needed ''From'' Davidson'''

'''LuxR expression cassette''' (promoter+RBS+LuxR+TT) '''James Barron''' 
(This is actually in the 07 & 08 REGISTRY! Part #I739003[08] and Part #'''I13018''' [07])

'''LuxI gene''' (RBS+LuxI)
(This is listed in the registry with a promoter Part#S03608[plated and tested SUCCESSFULLY]) 
'''Pallavi Penumetcha'''

'''lasR expression cassette''' (promoter+RBS+LasR+TT) '''James Barron''' 
(In progress...)

'''low copy amp vector''' (I150042 in I51020) '''Samantha''' (hopefully)

'''medium copy amp vector''' (I50032 in I51020) '''Samantha'''

l'''ow copy kan vector''' (not built yet) '''Samantha'''

'''medium copy kan vector''' (not built yet) '''Samantha'''

Davidson Projects with Updates

2008-07-07T17:36:28Z

JaBarron: /* Building LuxR receiver '''TESTER''' cell */

== Building LuxI sender '''TESTER''' cell ==
DONE! (ready to test) 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
DONE! (ready to test) 

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:K09100.jpg]] 
 

== Building LuxR constitutive protein ==
Promoter (pBad?) + [http://partsregistry.org/Part:BBa_ Fix this]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03954 Fix This]|| ? || ? || ? || ?
|-
|} 
[[Image:Const_LuxR.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|S03156|| Yes || Yes || Yes ||
|-
|B0015|| Yes|| Yes|| Yes ||
|-
|R0079|| Yes|| Yes || Yes ||
|-
|E0240|| Yes|| Yes || Yes ||
|-
|R0011|| Yes|| Yes|| Pending ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A
[[Image:S03954.jpg]] 
B
[[Image:S03956.jpg]] 
C
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A + Successful Ligation 1B 
Done! 
[[Image:K091118.jpg]] 
 
Ligation 3: R0011 + Ligation 2 

== Building LasR constitutive protein ==
Promoter (pBad? R0080) + [http://partsregistry.org/Part:BBa_S03954 S03954]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_R0080 R0080]|| ? || ? || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:Const_LasR.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|}

Davidson Projects with Updates

2008-07-07T17:36:08Z

JaBarron: /* Building LuxI sender '''TESTER''' cell */

== Building LuxI sender '''TESTER''' cell ==
DONE! (ready to test) 
{| border="1" cellpadding="2"
!width"50"|Part #
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03608 S03608]|| Yes || Yes || Ready || Successful
|-
|} 
[[Image:S03608.jpg]] 
 

== Building LuxR receiver '''TESTER''' cell ==
DONE! (ready to test) 

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_K09100 K09100]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:K09100.jpg]] 
 

== Building LuxR constitutive protein ==
Promoter (pBad?) + [http://partsregistry.org/Part:BBa_ Fix this]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_S03954 Fix This]|| ? || ? || ? || ?
|-
|} 
[[Image:Const_LuxR.jpg]]

== Building LasI sender '''TESTER''' cell ==
[http://partsregistry.org/Part:BBa_R0011 R0011] + [http://partsregistry.org/Part:BBa_B0034 B0034] + [http://partsregistry.org/Part:BBa_C0078 C0178]. 
[[Image:InsertHere.jpg]]
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|[http://partsregistry.org/Part:BBa_R0011 R0011]|| Yes || Yes || ||
|-
|[http://partsregistry.org/Part:BBa_B0034 B0034]|| Yes|| Yes || ||
|-
|[http://partsregistry.org/Part:BBa_C0178 C0178]|| Yes|| Yes || ||
|}
 

 

== Building LasR receiver '''TESTER''' cell ==
{| border="1" cellpadding="2"
!width="50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Successful Ligation?
!width="100"|Status of Testing the Device
|-
|S03156|| Yes || Yes || Yes ||
|-
|B0015|| Yes|| Yes|| Yes ||
|-
|R0079|| Yes|| Yes || Yes ||
|-
|E0240|| Yes|| Yes || Yes ||
|-
|R0011|| Yes|| Yes|| Pending ||
|-
|}
 

Ligation 1A S03156 + B0015 
Ligation 1B R0079 + E0240 
Ligation 1C B0015 + R0079 
Done! 
A
[[Image:S03954.jpg]] 
B
[[Image:S03956.jpg]] 
C
[[Image:C_1.jpg]]
 
 
Ligation 2: Successful Ligation 1A + Successful Ligation 1B 
Done! 
[[Image:K091118.jpg]] 
 
Ligation 3: R0011 + Ligation 2 

== Building LasR constitutive protein ==
Promoter (pBad? R0080) + [http://partsregistry.org/Part:BBa_S03954 S03954]

{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|[http://partsregistry.org/Part:BBa_R0080 R0080]|| ? || ? || ? || ?
|-
|[http://partsregistry.org/Part:BBa_S03954 S03954]|| Yes || Yes || Ready || Pending
|-
|} 
[[Image:Const_LasR.jpg]]

== Building LsrK/LsrR receiver '''TESTER''' cell ==
LsrKp + RBS + LsrK + TT

LsrRp + RBS + LsrR + reporter (GFP/RFP/YFP) + TT

LsrK needs to be constitutively expressed in order to receive the AI-2 signal. LsrR acts as its own repressor, so the reporter should not be activated until the AI-2 signal is received. Once LsrR binds to AI-2 molecule, the LsrR gene is activated and the positive feedback loop is initiated, which also turns on the reporter gene. 
{| border="1" cellpadding="2"
!width"50"|
!width="100"|Colonies from Registry Recovery?
!width="100"|Gel Verification?
!width="100"|Status of Testing the Device
!width="100"|Experimental Outcome
|-
|part no.|| ? || ? || ? || ?
|-
|} 

== Testing Crosstalk between Lux and Las ==
1) Mix LuxI sender cells with LasR receiver tester cells: negative control 
2) Mix LasI sender cells with LuxR receiver tester cells: negative control 
3) Mix LuxI sender cells with LuxR receiver tester cells: experiment we hope will glow green 
4) Mix LasI sender cells with LasR receiver tester cells: experiment we hope will glow green 
5) Verify LuxR receiver tester cells don't glow solo 
6) Verify LasR receiver tester cells don't glow solo 

== AND gate using pTetLac promoter ==
We have successfully built the pTetLac AND promoter: [http://partsregistry.org/Part:BBa_K091101 K091101]. 
This is sequence verified. 
Now we need the various LacI proteins (I12, X86 and I12+X86 variants). 
We need tetR repressor protein. We have tried to get it from the paper registry. 
We need to see if this AND gate works as designed. 
We need to test output to see if the two halves are balanced. 

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
 
<center>'''Davidson Approach''' 
'''Here is an idea Malcolm and Laurie developed.''' 
'''Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.''' </center>

<center> [[Image:XOR_AMC1b.jpg]] </center>
The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC6HSL])}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented [http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf by Egland and Greenberg]

'''Sequences we will need to make this XOR gate.''' 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Oligos
!width="50"|Oligo Design
|-
|lpLas'|| [[Image:pLasOligos.jpg]] || [[Image:pLas' oligos diagram.jpg]]
|-
|pLasXOR|| [[Image:pLasXOR Oligos.jpg]] || [[Image:pLasXOR.jpg]]
|-
|} 
{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward Primer
!width="50"|Reverse Primer
|-
|pLux|| [[Image:wtforward.jpg]]||[[Image:wtreverse.jpg]]
|-
|pLuxXOR||[[Image:XORforward.jpg]]||[[Image:XORreverse.jpg]]
|}

== Testing XOR Gate ==
We will need to have constitutive LasR and LuxR made in these cells. 
Then we will need to hit them with the two chemicals [http://partsregistry.org/AHL AHL types and commercial sources.]

== Making Better pLac promoter and LacI proteins ==
'''LacI wild-type gene sequence (ORF begins at the first amino acid)'''
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCATCA
ACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCG
CGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGC
TGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGT
GGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG
GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' 
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI_I12_X86 Mutation''' 
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA 
 
'''LacI Promoter''' 
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ Promoter''' 
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
'''LacIQ1 Promoter''' 
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG 
 
-These promoter sequences are taken from Glascock and Weickert 1998 

{| border="1" cellpadding="2"
!width="50"|Part
!width="50"|Forward
!width="50"|Reverse
|-
|lacIQ1 Promoter|| [[Image:placiq1_forward.jpg]] || [[Image:lacIq1reversecomplement.jpg]]
|-
|lacIQ Promoter|| [[Image:lacIqforwardprimer.jpg]] || [[Image:lacIqreversecomplement.jpg]]
|-
|LacI_I12||[[Image:LacI_I12forwardprimer.jpg]] || [[Image:LacI_I12reversecomplement.jpg]]
|-
|LacI|| [[Image:LacIforwardprimer.jpg]] || [[Image:LacIreversecomplement.jpg]]
|-
|LacI_X86 (Primers for step 1 of PCR)|| [[Image:LacIforwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|LacI_I12X86 (Primers for step 1 of PCR)|| [[Image:LacI_I12forwardprimer.jpg]] || [[Image:X86_X86I12reversecomplement.jpg]]
|-
|}

Needed From Davidson

2008-07-07T17:34:43Z

JaBarron:

'''Parts Needed From Davidson'''

LuxR expression cassette (promoter+RBS+LuxR+TT) James Barron 
(This is actually in the 07 & 08 REGISTRY! Part #I739003[08] and Part #I13018 [07])

LuxI gene (RBS+LuxI)
(This is listed in the registry with a promoter Part#S03608[plated and tested SUCCESSFULLY]) 
Pallavi Penumetcha

lasR expression cassette (promoter+RBS+LasR+TT) James Barron 
(In progress...)

low copy amp vector (I150042 in I51020) Samantha (hopefully)

medium copy amp vector (I50032 in I51020) Samantha

low copy kan vector (not built yet) Samantha

medium copy kan vector (not built yet) Samantha

Needed From Davidson

2008-07-07T15:54:44Z

JaBarron:

'''Parts Needed From Davidson'''

LuxR expression cassette (promoter+RBS+LuxR+TT) 
(This is actually in the 07 & 08 REGISTRY! Part #I739003[08] and Part #I13018 [07])

LuxI gene (RBS+LuxI)

LacI double mutant expression cassette (promoter+RBS+LacI+TT) 
Pallavi Penumetcha

lasR expression cassette (promoter+RBS+LasR+TT) 
James Barron (was sent off with MWSU last week Part #S03954)

low copy amp vector (I150042 in I51020) Samantha (hopefully)

medium copy amp vector (I50032 in I51020) Samantha

low copy kan vector (not built yet)

medium copy kan vector (not built yet)