View source for Davidson Projects with Updates

== Building LuxI sender '''TESTER''' cell ==
<BR>
{| 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
|-
|}<br>
[[Image:S03608.jpg]]<br>
<br>

== Building LuxR receiver '''TESTER''' cell ==
<br>
{| 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
|-
|}<br>
[[Image:K09100.jpg]]<br>
<br>

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

== 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 || ? || ? || ?
|}<br>
[[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].<br>
[[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 ||  ||
|}
<br>
[[Image:las.jpg]]
<br>

== 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 ||
|-
|}
<br>

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

== 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
|-
|}<br>
[[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.<br>
{| 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.|| ? || ? || ? || ?
|-
|}<br>

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

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

== Building XOR Gate ==
[http://partsregistry.org/AHL '''List of auto-inducers and their catalog numbers.''']
<br>
<center>'''Davidson Approach'''<br>
'''Here is an idea Malcolm and Laurie developed.''' <br>
'''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.'''<br></center>

<center> [[Image:XOR_AMC1b.jpg]]<br></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.''' <br>
{| 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]]
|-
|} <br>
{| 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.<br>
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 <br>
<br>
'''LacI_I12 Mutation (amino acid 3 is changed from CCA to TAT)''' <br>
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA <br>
<br>
'''LacI_X86 Mutation (amino acid 61 is changed from TCG to CTG)''' <br>
ATGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGC
GGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAA
ATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGC
AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCC
ATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGC
GCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATG
CTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCA
GGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG
CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA <br>
<br>
'''LacI_I12_X86 Mutation''' <br>
ATGAAATATGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAG
CTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGCTGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGAC
CAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATC
TGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG
AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT
CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACC
GCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT
TAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA<br>
<br>
'''LacI Promoter''' <br>
CGTTGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG <br>
<br>
'''LacIQ Promoter''' <br>
CGTTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGG <br>
<br>
'''LacIQ1 Promoter''' <br>
AGCGGCATGCATTTACGTTGACACCACCTTTCGCGGTATGGCATGATAGCGCCCGG <br>
<br>
-These promoter sequences are taken from Glascock and Weickert 1998<br>

{| 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]]
|-
|} <br>
'''Testing Lac promoters and proteins''' <br>
''Construct One'' <br>
[[Image:Construct12.jpg]] <br>
''Construct Two'' <br>
[[Image:Construct2.jpg]] <br>
-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.