GcatWiki - User contributions [en]

Primer Design for the RFP

2013-07-30T17:11:40Z

Spolpitiyaarachchige: Created page with "RFP for scaffold 2.0 Forward primer will bind to 5’ end of RFP JGGA2.0_RFP_for; GCATGGTCTCTGCGAGCTTCCTCCGAAGACGTTATC Reverse primer will to 3’ end of RFP JGGA2.0_RFP_r..."

RFP for scaffold 2.0

Forward primer will bind to 5’ end of RFP

JGGA2.0_RFP_for; GCATGGTCTCTGCGAGCTTCCTCCGAAGACGTTATC

Reverse primer will to 3’ end of RFP

JGGA2.0_RFP_rev; GCATGGTCTCAAGGCTTATTAAGCACCGGTGGAG

Primers for RFP Insertion between Junctions C and D

Oligos to be Ordered

JGGA2.0_RFP_for; GCATGGTCTCTGCGAGCTTCCTCCGAAGACGTTATC

JGGA2.0_RFP_rev; GCATGGTCTCAAGGCTTATTAAGCACCGGTGGAG

>BBa_E1010 Part-only sequence (706 bp)

Atg gct tcc tcc gaa gac gtt atc aaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcgaaatcgaaggtgaaggtg

aaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagta

cggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaacgtgttatgaacttcgaa

gacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtc

cggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaatcaaaatgcgtctgaaact

gaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaaaccgacatcaaactggac

atcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtca

ctccaccggtgcttaataa

Design of C dog oligos in J-GGA Scaffold for Scaffold 2.0

2013-07-30T17:09:46Z

Spolpitiyaarachchige: Created page with "C dog for scaffold 2.0 BD18 (medium) >BBa_J119024 Part-only sequence (88 bp) gggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaacatcttaatcatgcgacggagcgtttctaatg Oligo..."

C dog for scaffold 2.0

BD18 (medium)

>BBa_J119024 Part-only sequence (88 bp)

gggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaacatcttaatcatgcgacggagcgtttctaatg

Oligos to be Ordered

BD18_top; GGAGGGGCCCAAGTTCACTTAAAAAGGAGATCAACAATGAAAGCAATTTTCGTACTGAAACATCTTAATCATGCGACGGAGCG

BD18_bot; GCACCGCTCCGTCGCATGATTAAGATGTTTCAGTACGAAAATTGCTTTCATTGTTGATCTCCTTTTTAAGTGAACTTGGGCCC

Annealed

BD18_ GGAGGGGCCCAAGTTCACTTAAAAAGGAGATCAACAATGAAAGCAATTTTCGTACTGAAACATCTTAATCATGCGACGGAGCG

CCCGGGTTCAAGTGAATTTTTCCTCTAGTTGTTACTTTCGTTAAAAGCATGACTTTGTAGAATTAGTACGCTGCCTCGCCACG

J-GGA Promoter Sequence for Scaffold 2.0

2013-07-30T17:08:30Z

Spolpitiyaarachchige: Created page with "Promoter for scaffold 2.0 P5 highest J119031 BBa_J119031 Part-only sequence (36 bp) TTGACAATTAATCATCCGGCTCGTAATTTATGTGGA Oligos to be Ordered P5_top;GACCTTGACAATTAATCA..."

Promoter for scaffold 2.0

P5 highest J119031

BBa_J119031 Part-only sequence (36 bp)

TTGACAATTAATCATCCGGCTCGTAATTTATGTGGA

Oligos to be Ordered

P5_top;GACCTTGACAATTAATCATCCGGCTCGTAATTTATGTGGA

P5_Bottom;CCCATCCACATAAATTACGAGCCGGATGATTAATTGTCAA

Annealed

GACCTTGACAATTAATCATCCGGCTCGTAATTTATGTGGA

AACTGTTAATTAGTAGGCCGAGCATTAAATACACCTACCC

J-GGA Scaffold 2.0 Sequence

2013-07-30T17:07:33Z

Spolpitiyaarachchige:

Scaffold 2.0 Top

(5’to 3’)

AATTCGCGGCCGCTTCTAGAGCCGTATACACAGAGGACCACGTTGGGTCTTGAACCAGGAGCGGAGTGCTAATGACTGTGCGATTAGGCCTAACGAACAGACAGCTACTAGTAGCGGCCGCTGCA

Scaffold 2.0 Bottom

(5’to 3’)

GCGGCCGCTACTAGTAGCTGTCTGTTCGTTAGGCCTAATCGCACAGTCATTAGCACTCCGCTCCTGGTTCAAGACCCAACGTGGTCCTCTGTGTATACGGCTCTAGAAGCGGCCGCG

Annealed Scaffold Top and Bottom Oligos

Prefix Junction A Junction B Junction C Junction D suffix

AATTCGCGGCCGCTTCTAGAGCCGTATACACAGAGGACCACGTTGGGTCTTGAACCAGGAGCGGAGTGCTAATGACTGTGCGATTAGGCCTAACGAACAGACAGCTACTAGTAGCGGCCGCTGCA

GCGCCGGCGAAGATCTCGGCATATGTGTCTCCTGGTGCAACCCAGAACTTGGTCCTCGCCTCACGATTACTGACACGCTAATCCGGATTGCTTGTCTGTCGATGATCATCGCCGGCG

Scaffold 2.0 Ordering

Scaffold2.0_Top1; AATTCGCGGCCGCTTCTAGAGCCGTATACACAGAGGACCACGTTGGG

Scaffold2.0_Top2; TCTTGAACCAGGAGCGGAGTGCTAATGACTGTGCGATTAGGCCTAACGAACAGACAGCTACTAGTAGCGGCCGCTGCA

Scaffold2.0_Bottom1; GCGGCCGCTACTAGTAGCTGTCTGTTCGTTAGGCCTAATCGCAC

Scaffold2.0_Bottom2; AGTCATTAGCACTCCGCTCCTGGTTCAAGACCCAACGTGGTCCTCTGTGTATACGGCTCTAGAAGCGGCCGCG

J-GGA Scaffold 2.0 Primer

2013-07-30T17:06:39Z

Spolpitiyaarachchige: Created page with "Scaffold 2.0 Primers JunctionA_forward; GCATGGTCTCTCCGTATACACAGAGGACC JunctionA_reverse; GCATGGTCTCTGGTCCTCTGTGTATACGG JunctionB_forward; GCATGGTCTCTTGGGTCTTGAACCAGGAG Ju..."

Scaffold 2.0 Primers

JunctionA_forward; GCATGGTCTCTCCGTATACACAGAGGACC

JunctionA_reverse; GCATGGTCTCTGGTCCTCTGTGTATACGG

JunctionB_forward; GCATGGTCTCTTGGGTCTTGAACCAGGAG

JunctionB_reverse; GCATGGTCTCTCTCCTGGTTCAAGACCCA

JunctionC_forward; GCATGGTCTCTGTGCTAATGACTGTGCGA

JunctionC_reverse; GCATGGTCTCTTCGCACAGTCATTAGCAC

JunctionD_forward; GCATGGTCTCTGCCTAACGAACAGACAGC

JunctionD_reverse; GCATGGTCTCTGCTGTCTGTTCGTTAGGC

J-GGA Scaffold 2.0 Sequence

2013-07-30T17:04:57Z

Spolpitiyaarachchige: Created page with "Scaffold 2.0 Top (5’to 3’) AATTCGCGGCCGCTTCTAGAGCCGTATACACAGAGGACCACGTTGGGTCTTGAACCAGGAGCGGAGTGCTAATGACTGTGCGATTAGGCCTAACGAACAGACAGCTACTAGTAGCGGCCGCTGCA Scaffold 2.0 Bo..."

J-GGA Scaffold 2.0 Design

2013-07-30T17:01:52Z

Spolpitiyaarachchige:

[[J-GGA Scaffold 2.0 Sequence]]

[[J-GGA Scaffold 2.0 Primer]]

[[J-GGA Promoter Sequence for Scaffold 2.0]]

[[Design of C dog oligos in J-GGA Scaffold for Scaffold 2.0]]

[[Primer Design for the RFP]]

J-GGA Scaffold 2.0 Design

2013-07-30T17:00:11Z

Spolpitiyaarachchige: Created page with "J-GGA Scaffold Sequence J-GGA Scaffold Primer J-GGA Promoter Sequence Design of C dog oligos in J-GGA Scaffold Primer Design for the RFP"

[[J-GGA Scaffold Sequence]]

[[J-GGA Scaffold Primer]]

[[J-GGA Promoter Sequence]]

[[Design of C dog oligos in J-GGA Scaffold]]

[[Primer Design for the RFP]]

Biology

2013-07-30T16:58:35Z

Spolpitiyaarachchige:

[[Davidson Protocols]] 
[[MWSU_protocols]] 

[[J-GGA Scaffold Design]]

[[J-GGA Scaffold 2.0 Design]]

[[Origin of Replication]]

[[File:Gelpouring.jpg|200px|thumb|right|Look, Mom, no EtBr!]]

[//gcat.davidson.edu/SynBio13/primer GGA Primer Designer]

'''Topics to Investigate'''

'''Family of Xanthine compounds'''

[[File:Caffeine_demethylase_word_doc.docx]]

High Priority = more people

* '''Phoebe and Claire:''' eCDM8 working to make theophylline with biosensor and fitness modules (TetR) to report out. 
Which plasmid carries which device? 
Biosensor 
Fitness (tetA) 
feeds into stress module 
* '''Sachii and Jess:''' Determine the junction sequences and associated primers 
(use existing rules) 
build scaffold for insert segments 
include orI (collaborate with copy number research) 
include drug resistance gene 
test JGG design 
* '''Spencer and Sara:''' test fitness module with adhE
compare with tetA 
fine tune tetA resistance 
combine with tetA 
Does theophylline diffuse across the membrane and thus the system fails? 
* '''Brandon and Erich:''' Copy number of plasmids 
Can we alter Ori to produce a range of plasmid densities in ''E. coli''? 

Second Order Priority = one person
* produce different CDM alleles
* swap out different components (promoters, RBS, alleles)
* test out programmed evolution
* Stress module needs more work (see eCDM8 outputs)

Biology

2013-07-30T16:56:23Z

Spolpitiyaarachchige:

[[Davidson Protocols]] 
[[MWSU_protocols]] 

[[J-GGA Scaffold 1.0 Design]]

[[J-GGA Scaffold 2.0 Design]]

[[Origin of Replication]]

[[File:Gelpouring.jpg|200px|thumb|right|Look, Mom, no EtBr!]]

[//gcat.davidson.edu/SynBio13/primer GGA Primer Designer]

'''Topics to Investigate'''

'''Family of Xanthine compounds'''

[[File:Caffeine_demethylase_word_doc.docx]]

High Priority = more people

* '''Phoebe and Claire:''' eCDM8 working to make theophylline with biosensor and fitness modules (TetR) to report out. 
Which plasmid carries which device? 
Biosensor 
Fitness (tetA) 
feeds into stress module 
* '''Sachii and Jess:''' Determine the junction sequences and associated primers 
(use existing rules) 
build scaffold for insert segments 
include orI (collaborate with copy number research) 
include drug resistance gene 
test JGG design 
* '''Spencer and Sara:''' test fitness module with adhE
compare with tetA 
fine tune tetA resistance 
combine with tetA 
Does theophylline diffuse across the membrane and thus the system fails? 
* '''Brandon and Erich:''' Copy number of plasmids 
Can we alter Ori to produce a range of plasmid densities in ''E. coli''? 

Second Order Priority = one person
* produce different CDM alleles
* swap out different components (promoters, RBS, alleles)
* test out programmed evolution
* Stress module needs more work (see eCDM8 outputs)

Biology

2013-06-11T15:51:51Z

Spolpitiyaarachchige:

[[Davidson Protocols]] 
[[MWSU_protocols]] 

[[J-GGA Scaffold Design]]

[[Origin of Replication]]

[[File:Gelpouring.jpg|200px|thumb|right|Look, Mom, no EtBr!]]

'''Topics to Investigate'''

High Priority = more people

* '''Phoebe and Claire:''' eCDM8 working to make theophylline with biosensor and fitness modules (TetR) to report out. 
Which plasmid carries which device? 
Biosensor 
Fitness (tetA) 
feeds into stress module 
* '''Sachii and Jess:''' Determine the junction sequences and associated primers 
(use existing rules) 
build scaffold for insert segments 
include orI (collaborate with copy number research) 
include drug resistance gene 
test JGG design 
* '''Spencer and Sara:''' test fitness module with adhE
compare with tetA 
fine tune tetA resistance 
combine with tetA 
Does theophylline diffuse across the membrane and thus the system fails? 
* '''Brandon and Erich:''' Copy number of plasmids 
Can we alter Ori to produce a range of plasmid densities in ''E. coli''? 

Second Order Priority = one person
* produce different CDM alleles
* swap out different components (promoters, RBS, alleles)
* test out programmed evolution
* Stress module needs more work (see eCDM8 outputs)

Primer Design for the eCDM8 and RFP

2013-06-11T15:51:08Z

Spolpitiyaarachchige:

PCR primers for insertion of Genes between Junctions C and D

1. Conduct Inverse PCR using scaffold with promoters and C dogs and J-GGA_C_rev and J-GGA_D_for.

2. Conduct PCR of eCDM8 gene and RFP gene templates with primers below.

3. Conduct J-GGA with products from 1 and 2 above.

Gene primer considerations.

1. Four extra bases on 5’ end (eg. GCAT)

2. BsaI site directed toward the gene

3. Sticky end of 5’ CATC 3’ for Junction C on For primer

4. Sticky end of 5’ TCCG 3’ for Junction D on Rev primer

5. 18 nt of gene coding sequence on each primer

Primer designs for the eCDM8 gene

Forward ECDM8 gcatggtctcacatcaaagaaatgccgcagccg

Rev ECDM8 ccagggtctcatccgttattaacctgcccaaac

5’ gttTgggcaggttaataa 3’

3’ caaacccgtccaattatt 5’

>BBa_J100100 Part-only sequence (3204 bp)

gaattcgcggccgcttctagagggtctcgaatgaccatcaaagaaatgccgcagccgaaaacctttggtgaactgaaaaatctgccgctgctgaataccg

ataaaccggttcatgcactgatgaaaattgcagatgaactgggcgagatctttaaattcgaagcaccgggtagcgttacccgttatctgagcagccagcg

tctgattaaagaagcatgcgacgaaagccgctttgataaaaatctgattcagtggctgaaattcatccgcgattttctgggtgatggtctgagcaccagc

tggacccatgaaaaaaactggaaaaaagcccataatattctgctgccgagctttagccagcaggcaatgaaaggttatcatgcaatgatggttgatattg

ccgttcagctggttcagaaatgggaacgtctgaatgcagatgaacatattgaagttccggaagatatgacccgtctgaccctggataccattggtctgtg

tggttttaactatcgcttcaacagcttttatcgtgatcagccgcatccgtttgttaccagcatggttcgtgcactggatgaagcagtgaataaatggcag

cgtgcaaatccggatgatccggcatatgatgaaaataaacgtcagttccaagaggatattaaagtgatgaatgacctggtggacaaaattatcgcagatc

gtaaagcaagcggtgaacagagtgatgatctgctgacccatatgctgaatggtaaagatccggaaaccggtgaaccgctggatgatgaaaacattcgtta

tcagattattaccttcctgattgccggtcatgaaaccaccagtggtctgctgagctttgcactgtattttctggttaaaaatccgcacgttctgcaaaaa

gcagccgaagaagcagcacgcgttctggttgatccggttccgagctataaacaggttaaacagctgaaatatgtgggcatggttctgaatgaagcactgc

gtctgtggccgacctttccgtggtttagcctgtatgcaaaagaagataccgttctgggaggtgaatatccgctggaaaaaggtgatgaactgatggttct

gattccgcagctgcatcgtgataaaaccatttggggtgatgatgtggaagaatttcgtccggaacgttttgaaaatccgagcgcaattccgcagcatgcc

tttaaaccgtttggtaatggtcagcgtgcatgtattggtcagcagtttgcactgcatgaagccaccctggttctgggtatgatgctgaaacattttgatt

ttgaggaccacaccaactatgagctggatattaaaggcaccctgaccctgaaaccggaaggttttgttgttaaagccaaaagcaaaaaaatcccgctggg

tggtattccgagcccgagcaccgaacagagcgcaaaaaaagttcgtaaaaaagccgaaaacgcccataatacaccgctgctggttctgtatggtagcaat

atgggtacagccgaaggcaccgcacgtgatctggcagatattgcaatgagcaaaggttttgcaccgcaggttgcaaccctggatagccatgcaggtaatc

tgcctcgtgaaggtgcagttctgattgttaccgcaagctataatggtcatccgcctgataatgcaaaacagtttgttgattggctggatcaggcaagcgc

agatgaagttaaaggtgttcgttatagcgtttttggttgcggtgataaaaactgggcaaccacctatcagaaagttccggcatttattgatgaaaccctg

gcagcaaaaggtgcagaaaatattgcagatcgtggtgaaaccgatgccagtgatgattttgaaggcacctatgaagaatggcgtgaacatatgtggtcag

atgttgcagcctattttaacctggatatcgaaaacagcgaggacaataaaagcaccctgagcctgcaatttgtggatagcgcagcagatatgccgctggc

aaaaatgcatggtgcatttagcaccaatgttgttgcaagcaaagaactgcaacagcctggtagcgcacgtagcacccgtcatctggaaattgaactgccg

aaagaagcaagctatcaagagggtgatcatctgggtgttattccgcgtaattatgaaggtattgttaatcgtgttaccgcacgttttggtctggatgcaa

gccagcagattcgtctggaagcagaagaagaaaaactggcacatctgccgctggccaaaaccgttagcgttgaagaactgctgcaatatgttgaactgca

agatccggttacccgtacccagctgcgtgcaatggcagcaaaaaccgtttgtccgcctcataaagttgaactggaagcactgctggagaaacaggcatat

aaagaacaggttctggcaaaacgtctgaccatgctggaactgctggaaaaatatccggcatgcgaaatgaaattcagcgaatttattgcactgctgccga

gtattcgtccgcgttattatagcattagcagcagtccgcgtgttgatgaaaaacaggccagcattaccgttagcgtggttagcggtgaagcatggtcagg

ttatggtgaatataaaggtattgccagcaattatctggccgaactgcaagaaggtgataccattacctgttttattagcacaccgcagagcgaatttacc

ctgccgaaagatcctgaaacaccgctgattatggttggtccgggtacaggtgttgcaccgtttcgtggttttgttcaggcacgtaaacaactgaaagaac

agggtcagagcctgggtgaagcacatctgtattttggttgtcgtagtccgcatgaggattatctgtatcaagaagaactggaaaacgcacagagcgaagg

tattattaccctgcataccgcatttagccgtatgccgaatcagccgaaaacatatgttcagcatgttatggaacaggatggcaaaaaactgattgaactg

ctggatcagggtgcccatttctatatttgtggtgatggtagccagatggcaccggcagttgaagcaaccctgatgaaaagctatgcagatgttcatcagg

ttagcgaagcagatgcacgtctgtggctgcaacagctggaagaaaaaggtcgttatgcaaaagatgtttgggcaggttaataatactagtagcggccgct
gcag

Primer designs for the RFP gene

RFP Forward gcatggtctcgcatcgcttcctccgaagacgtt

RFP Rev ccagggtctcatccgttattaagcaccggtgga

5’ Tccaccggtgcttaataa 3’

3’ Aggtggccacgaattatt 5’

RFP Gene Sequence

>BBa_E1010 Part-only sequence (706 bp)

atggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcgaaatcgaaggtgaaggtg

aaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagta

cggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaacgtgttatgaacttcgaa

gacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtc

cggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaatcaaaatgcgtctgaaact

gaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaaaccgacatcaaactggac

atcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgcttaataa

Primer Design for the eCDM8 and RFP

2013-06-11T15:50:42Z

Spolpitiyaarachchige:

PCR primers for insertion of Genes between Junctions C and D

1. Conduct Inverse PCR using scaffold with promoters and C dogs and J-GGA_C_rev and J-GGA_D_for.

2. Conduct PCR of eCDM8 gene and RFP gene templates with primers below.

3. Conduct J-GGA with products from 1 and 2 above.

Gene primer considerations.

1. Four extra bases on 5’ end (eg. GCAT)

2. BsaI site directed toward the gene

3. Sticky end of 5’ CATC 3’ for Junction C on For primer

4. Sticky end of 5’ TCCG 3’ for Junction D on Rev primer

5. 18 nt of gene coding sequence on each primer

Primer designs for the eCDM8 gene

Forward ECDM8 gcatggtctcacatcaaagaaatgccgcagccg

Rev ECDM8 ccagggtctcatccgttattaacctgcccaaac

5’ gttTgggcaggttaataa 3’

3’ caaacccgtccaattatt 5’

>BBa_J100100 Part-only sequence (3204 bp)
gaattcgcggccgcttctagagggtctcgaatgaccatcaaagaaatgccgcagccgaaaacctttggtgaactgaaaaatctgccgctgctgaataccg

ataaaccggttcatgcactgatgaaaattgcagatgaactgggcgagatctttaaattcgaagcaccgggtagcgttacccgttatctgagcagccagcg

tctgattaaagaagcatgcgacgaaagccgctttgataaaaatctgattcagtggctgaaattcatccgcgattttctgggtgatggtctgagcaccagc

tggacccatgaaaaaaactggaaaaaagcccataatattctgctgccgagctttagccagcaggcaatgaaaggttatcatgcaatgatggttgatattg

ccgttcagctggttcagaaatgggaacgtctgaatgcagatgaacatattgaagttccggaagatatgacccgtctgaccctggataccattggtctgtg

tggttttaactatcgcttcaacagcttttatcgtgatcagccgcatccgtttgttaccagcatggttcgtgcactggatgaagcagtgaataaatggcag

cgtgcaaatccggatgatccggcatatgatgaaaataaacgtcagttccaagaggatattaaagtgatgaatgacctggtggacaaaattatcgcagatc

gtaaagcaagcggtgaacagagtgatgatctgctgacccatatgctgaatggtaaagatccggaaaccggtgaaccgctggatgatgaaaacattcgtta

tcagattattaccttcctgattgccggtcatgaaaccaccagtggtctgctgagctttgcactgtattttctggttaaaaatccgcacgttctgcaaaaa

gcagccgaagaagcagcacgcgttctggttgatccggttccgagctataaacaggttaaacagctgaaatatgtgggcatggttctgaatgaagcactgc

gtctgtggccgacctttccgtggtttagcctgtatgcaaaagaagataccgttctgggaggtgaatatccgctggaaaaaggtgatgaactgatggttct

gattccgcagctgcatcgtgataaaaccatttggggtgatgatgtggaagaatttcgtccggaacgttttgaaaatccgagcgcaattccgcagcatgcc

tttaaaccgtttggtaatggtcagcgtgcatgtattggtcagcagtttgcactgcatgaagccaccctggttctgggtatgatgctgaaacattttgatt

ttgaggaccacaccaactatgagctggatattaaaggcaccctgaccctgaaaccggaaggttttgttgttaaagccaaaagcaaaaaaatcccgctggg

tggtattccgagcccgagcaccgaacagagcgcaaaaaaagttcgtaaaaaagccgaaaacgcccataatacaccgctgctggttctgtatggtagcaat

atgggtacagccgaaggcaccgcacgtgatctggcagatattgcaatgagcaaaggttttgcaccgcaggttgcaaccctggatagccatgcaggtaatc

tgcctcgtgaaggtgcagttctgattgttaccgcaagctataatggtcatccgcctgataatgcaaaacagtttgttgattggctggatcaggcaagcgc

agatgaagttaaaggtgttcgttatagcgtttttggttgcggtgataaaaactgggcaaccacctatcagaaagttccggcatttattgatgaaaccctg

gcagcaaaaggtgcagaaaatattgcagatcgtggtgaaaccgatgccagtgatgattttgaaggcacctatgaagaatggcgtgaacatatgtggtcag

atgttgcagcctattttaacctggatatcgaaaacagcgaggacaataaaagcaccctgagcctgcaatttgtggatagcgcagcagatatgccgctggc

aaaaatgcatggtgcatttagcaccaatgttgttgcaagcaaagaactgcaacagcctggtagcgcacgtagcacccgtcatctggaaattgaactgccg

aaagaagcaagctatcaagagggtgatcatctgggtgttattccgcgtaattatgaaggtattgttaatcgtgttaccgcacgttttggtctggatgcaa

gccagcagattcgtctggaagcagaagaagaaaaactggcacatctgccgctggccaaaaccgttagcgttgaagaactgctgcaatatgttgaactgca

agatccggttacccgtacccagctgcgtgcaatggcagcaaaaaccgtttgtccgcctcataaagttgaactggaagcactgctggagaaacaggcatat

aaagaacaggttctggcaaaacgtctgaccatgctggaactgctggaaaaatatccggcatgcgaaatgaaattcagcgaatttattgcactgctgccga

gtattcgtccgcgttattatagcattagcagcagtccgcgtgttgatgaaaaacaggccagcattaccgttagcgtggttagcggtgaagcatggtcagg

ttatggtgaatataaaggtattgccagcaattatctggccgaactgcaagaaggtgataccattacctgttttattagcacaccgcagagcgaatttacc

ctgccgaaagatcctgaaacaccgctgattatggttggtccgggtacaggtgttgcaccgtttcgtggttttgttcaggcacgtaaacaactgaaagaac

agggtcagagcctgggtgaagcacatctgtattttggttgtcgtagtccgcatgaggattatctgtatcaagaagaactggaaaacgcacagagcgaagg

tattattaccctgcataccgcatttagccgtatgccgaatcagccgaaaacatatgttcagcatgttatggaacaggatggcaaaaaactgattgaactg

ctggatcagggtgcccatttctatatttgtggtgatggtagccagatggcaccggcagttgaagcaaccctgatgaaaagctatgcagatgttcatcagg

ttagcgaagcagatgcacgtctgtggctgcaacagctggaagaaaaaggtcgttatgcaaaagatgtttgggcaggttaataatactagtagcggccgct
gcag

Primer designs for the RFP gene

RFP Forward gcatggtctcgcatcgcttcctccgaagacgtt

RFP Rev ccagggtctcatccgttattaagcaccggtgga

5’ Tccaccggtgcttaataa 3’

3’ Aggtggccacgaattatt 5’

RFP Gene Sequence

>BBa_E1010 Part-only sequence (706 bp)

atggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcgaaatcgaaggtgaaggtg

aaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagta

cggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaacgtgttatgaacttcgaa

gacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtc

cggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaatcaaaatgcgtctgaaact

gaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaaaccgacatcaaactggac

atcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgcttaataa

Primer Design for the eCDM8 and RFP

2013-06-11T15:49:53Z

Spolpitiyaarachchige: Created page with "PCR primers for insertion of Genes between Junctions C and D 1. Conduct Inverse PCR using scaffold with promoters and C dogs and J-GGA_C_rev and J-GGA_D_for. 2. Conduct PCR ..."

PCR primers for insertion of Genes between Junctions C and D

1. Conduct Inverse PCR using scaffold with promoters and C dogs and J-GGA_C_rev and J-GGA_D_for.

2. Conduct PCR of eCDM8 gene and RFP gene templates with primers below.

3. Conduct J-GGA with products from 1 and 2 above.

Gene primer considerations.

1. Four extra bases on 5’ end (eg. GCAT)

2. BsaI site directed toward the gene

3. Sticky end of 5’ CATC 3’ for Junction C on For primer

4. Sticky end of 5’ TCCG 3’ for Junction D on Rev primer

5. 18 nt of gene coding sequence on each primer

Primer designs for the eCDM8 gene

Forward ECDM8 gcatggtctcacatcaaagaaatgccgcagccg

Rev ECDM8 ccagggtctcatccgttattaacctgcccaaac

5’ gttTgggcaggttaataa 3’

3’ caaacccgtccaattatt 5’

>BBa_J100100 Part-only sequence (3204 bp)
gaattcgcggccgcttctagagggtctcgaatgaccatcaaagaaatgccgcagccgaaaacctttggtgaactgaaaaatctgccgctgctgaataccg

ataaaccggttcatgcactgatgaaaattgcagatgaactgggcgagatctttaaattcgaagcaccgggtagcgttacccgttatctgagcagccagcg

tctgattaaagaagcatgcgacgaaagccgctttgataaaaatctgattcagtggctgaaattcatccgcgattttctgggtgatggtctgagcaccagc

tggacccatgaaaaaaactggaaaaaagcccataatattctgctgccgagctttagccagcaggcaatgaaaggttatcatgcaatgatggttgatattg

ccgttcagctggttcagaaatgggaacgtctgaatgcagatgaacatattgaagttccggaagatatgacccgtctgaccctggataccattggtctgtg

tggttttaactatcgcttcaacagcttttatcgtgatcagccgcatccgtttgttaccagcatggttcgtgcactggatgaagcagtgaataaatggcag

cgtgcaaatccggatgatccggcatatgatgaaaataaacgtcagttccaagaggatattaaagtgatgaatgacctggtggacaaaattatcgcagatc

gtaaagcaagcggtgaacagagtgatgatctgctgacccatatgctgaatggtaaagatccggaaaccggtgaaccgctggatgatgaaaacattcgtta

tcagattattaccttcctgattgccggtcatgaaaccaccagtggtctgctgagctttgcactgtattttctggttaaaaatccgcacgttctgcaaaaa

gcagccgaagaagcagcacgcgttctggttgatccggttccgagctataaacaggttaaacagctgaaatatgtgggcatggttctgaatgaagcactgc

gtctgtggccgacctttccgtggtttagcctgtatgcaaaagaagataccgttctgggaggtgaatatccgctggaaaaaggtgatgaactgatggttct

gattccgcagctgcatcgtgataaaaccatttggggtgatgatgtggaagaatttcgtccggaacgttttgaaaatccgagcgcaattccgcagcatgcc

tttaaaccgtttggtaatggtcagcgtgcatgtattggtcagcagtttgcactgcatgaagccaccctggttctgggtatgatgctgaaacattttgatt

ttgaggaccacaccaactatgagctggatattaaaggcaccctgaccctgaaaccggaaggttttgttgttaaagccaaaagcaaaaaaatcccgctggg

tggtattccgagcccgagcaccgaacagagcgcaaaaaaagttcgtaaaaaagccgaaaacgcccataatacaccgctgctggttctgtatggtagcaat

atgggtacagccgaaggcaccgcacgtgatctggcagatattgcaatgagcaaaggttttgcaccgcaggttgcaaccctggatagccatgcaggtaatc

tgcctcgtgaaggtgcagttctgattgttaccgcaagctataatggtcatccgcctgataatgcaaaacagtttgttgattggctggatcaggcaagcgc

agatgaagttaaaggtgttcgttatagcgtttttggttgcggtgataaaaactgggcaaccacctatcagaaagttccggcatttattgatgaaaccctg

gcagcaaaaggtgcagaaaatattgcagatcgtggtgaaaccgatgccagtgatgattttgaaggcacctatgaagaatggcgtgaacatatgtggtcag

atgttgcagcctattttaacctggatatcgaaaacagcgaggacaataaaagcaccctgagcctgcaatttgtggatagcgcagcagatatgccgctggc

aaaaatgcatggtgcatttagcaccaatgttgttgcaagcaaagaactgcaacagcctggtagcgcacgtagcacccgtcatctggaaattgaactgccg

aaagaagcaagctatcaagagggtgatcatctgggtgttattccgcgtaattatgaaggtattgttaatcgtgttaccgcacgttttggtctggatgcaa

gccagcagattcgtctggaagcagaagaagaaaaactggcacatctgccgctggccaaaaccgttagcgttgaagaactgctgcaatatgttgaactgca

agatccggttacccgtacccagctgcgtgcaatggcagcaaaaaccgtttgtccgcctcataaagttgaactggaagcactgctggagaaacaggcatat

aaagaacaggttctggcaaaacgtctgaccatgctggaactgctggaaaaatatccggcatgcgaaatgaaattcagcgaatttattgcactgctgccga

gtattcgtccgcgttattatagcattagcagcagtccgcgtgttgatgaaaaacaggccagcattaccgttagcgtggttagcggtgaagcatggtcagg

ttatggtgaatataaaggtattgccagcaattatctggccgaactgcaagaaggtgataccattacctgttttattagcacaccgcagagcgaatttacc

ctgccgaaagatcctgaaacaccgctgattatggttggtccgggtacaggtgttgcaccgtttcgtggttttgttcaggcacgtaaacaactgaaagaac

agggtcagagcctgggtgaagcacatctgtattttggttgtcgtagtccgcatgaggattatctgtatcaagaagaactggaaaacgcacagagcgaagg

tattattaccctgcataccgcatttagccgtatgccgaatcagccgaaaacatatgttcagcatgttatggaacaggatggcaaaaaactgattgaactg

ctggatcagggtgcccatttctatatttgtggtgatggtagccagatggcaccggcagttgaagcaaccctgatgaaaagctatgcagatgttcatcagg

ttagcgaagcagatgcacgtctgtggctgcaacagctggaagaaaaaggtcgttatgcaaaagatgtttgggcaggttaataatactagtagcggccgct
gcag

Primer designs for the RFP gene

RFP Forward gcatggtctcgcatcgcttcctccgaagacgtt

RFP Rev ccagggtctcatccgttattaagcaccggtgga

5’ Tccaccggtgcttaataa 3’

3’ Aggtggccacgaattatt 5’

RFP Gene Sequence

>BBa_E1010 Part-only sequence (706 bp)

atggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcgaaatcgaaggtgaaggtg

aaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagta

cggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaacgtgttatgaacttcgaa

gacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtc

cggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaatcaaaatgcgtctgaaact

gaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaaaccgacatcaaactggac

atcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgcttaataa

Design of C dog oligos in J-GGA Scaffold

2013-06-11T15:47:01Z

Spolpitiyaarachchige: Created page with "Design of C dog oligos in J-GGA Scaffold C dogs Used BD2 (lowest) >BBa_J119025 Part-only sequence (88 bp) gggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaacatcttaat..."

Design of C dog oligos in J-GGA Scaffold

C dogs Used

BD2 (lowest)

>BBa_J119025 Part-only sequence (88 bp)

gggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaacatcttaatcatgctaaggaggttttctaatg

BD18 (medium)

>BBa_J119024 Part-only sequence (88 bp)

gggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaacatcttaatcatgcgacggagcgtttctaatg

Aligned

BD2 gggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaacatcttaatcatgctaaggaggttttctaatg

BD18 Gggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaacatcttaatcatgcgacggagcgtttctaatg

Cons Gggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaacatcttaatcatgckacggagsktttctaatg

Number of Sequences = 2 power 3 = 8

New J-GGA Scaffold (after PCR mutagenesis with above primers)

Prefix: Gaattcgcggccgcttctagag

Junction A: Atagtctgttcatggtgc

Spacer: acgt

Junction B: ggctgatgtagttaaggc

Spacer: cgga

Junction C: gttctaatggggaccatc

Spacer: ttag

Junction D: cggagctatgtattcctg

Suffix: tactagtagcggccgctgcag

Sticky ends needed:

Junction B: AGGC

Junction C: GTTC

Oligos to be Ordered

BD2_top; AGGCgggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaacatcttaatcatgctaaggaggt

BD2_bot; GAACACCTCCTTAGCATGATTAAGATGTTTCAGTACGAAAATTGCTTTCATTGTTGATCTCCTTTTTAAGTGAACTTGGGCCC

BD2_18Cons_Top;

AGGCGggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaacatcttaatcatgckacggagsk

BD2_18Cons_Top;

GAACMSCTCCGTMGCATGATTAAGATGTTTCAGTACGAAAATTGCTTTCATTGTTGATCTCCTTTTTAAGTGAACTTGGGCCC

Annealed

BD2_ AGGCGGGCCCAAGTTCACTTAAAAAGGAGATCAACAATGAAAGCAATTTTCGTACTGAAACATCTTAATCATGCTAAGGAGGT

CCCGGGTTCAAGTGAATTTTTCCTCTAGTTGTTACTTTCGTTAAAAGCATGACTTTGTAGAATTAGTACGATTCCTCCACAAG

BD2_18CONS

AGGCGGGCCCAAGTTCACTTAAAAAGGAGATCAACAATGAAAGCAATTTTCGTACTGAAACATCTTAATCATGCKACGGAGSK

CCCGGGTTCAAGTGAATTTTTCCTCTAGTTGTTACTTTCGTTAAAAGCATGACTTTGTAGAATTAGTACGMTGCCTCSMCAAG

J-GGA Promoter Sequence

2013-06-11T15:44:45Z

Spolpitiyaarachchige: Created page with "Design of promoters for use in J-GGA Scaffold Promoters Used P5 highest J119031 BBa_J119031 Part-only sequence (36 bp) ttgacaattaatcatccggctcgtaatttatgtgga P14 medium J11..."

Design of promoters for use in J-GGA Scaffold

Promoters Used

P5 highest J119031

BBa_J119031 Part-only sequence (36 bp)

ttgacaattaatcatccggctcgtaatttatgtgga

P14 medium J119033

BBa_J119033 Part-only sequence (36 bp)

ttgacaattaatcatccggctcgtataatgtgtgga

P9 lower J119032

BBa_J119032 Part-only sequence (35 bp)

ttgcctcttaatcatcggctcgtataatgtgtgga

Aligned (J31,32, and 33)

BBa_J119031 ttgacaattaatcatccggctcgtaatttatgtgga

BBa_J119033 ttgacaattaatcatccggctcgtataatgtgtgga

BBa_J119032 ttgcctcttaatcatcggctcgtatwaatgtgtgga

Consensus TTGMCWMTTAATCATCSGSYYSKWWWWWTRTGTGGA

Number of Sequences = 2 power 15 = 32,768

Aligned (J31 and 32 only)

BBa_J119031 ttgacaattaatcatccggctcgtaatttatgtgga

BBa_J119033 ttgacaattaatcatccggctcgtataatgtgtgga

Consensus TTGACAATTAATCATCCGGCTCGTAWWWTRTGTGGA

Number of Sequences = 2 power 4 = 16

New J-GGA Scaffold (after PCR mutagenesis with above primers)

Prefix: Gaattcgcggccgcttctagag

Junction A: Atagtctgttcatggtgc

Spacer: acgt

Junction B: ggctgatgtagttaaggc

Spacer: cgga

Junction C: gttctaatggggaccatc

Spacer: ttag

Junction D: cggagctatgtattcctg

Suffix: tactagtagcggccgctgcag

Sticky ends needed:

Junction A: GTGC

Junction B: GGCT

BBA_J31 GTGCTTGACAATTAATCATCCGGCTCGTAATTTATGTGGA

AACTGTTAATTAGTAGGCCGAGCATTAAATACACCTTCGG

BBA_J33 GTGCTTGACAATTAATCATCCGGCTCGTATAATGTGTGGA

AACTGTTAATTAGTAGGCCGAGCATATTACACACCTTCGG

CONS GTGCTTGACAATTAATCATCCGGCTCGTAWWWTRTGTGGA

AACTGTTAATTAGTAGGCCGAGCATWWWAYACACCTTCGG

Oligos to be Ordered

J31top;GTGCTTGACAATTAATCATCCGGCTCGTAATTTATGTGGA

J31bot; GGCTTCCACATAAATTACGAGCCGGATGATTAATTGTCAA

J33top; GTGCTTGACAATTAATCATCCGGCTCGTATAATGTGTGGA

J33bot; GGCTTCCACACATTATACGAGCCGGATGATTAATTGTCAA

J31+32top; GTGCTTGACAATTAATCATCCGGCTCGTAWWWTRTGTGGA

J31+32bot; GGCTTCCACAYAWWWTACGAGCCGGATGATTAATTGTCAA

J-GGA Scaffold Primer

2013-06-11T15:43:59Z

Spolpitiyaarachchige: Created page with "PRIMERS TO BE USED FOR INVERTED PCR OF SCAFFOLD J-GGA_A_rev (29 nt) GCATGGTCTCAGCACCATGAACAGACTAT J-GGA_B_for (29 nt) CCAGGGTCTCTGGCTGATGTAGTTAAGGC J-GGA_B_rev (29 nt) ..."

PRIMERS TO BE USED FOR INVERTED PCR OF SCAFFOLD

J-GGA_A_rev (29 nt)

GCATGGTCTCAGCACCATGAACAGACTAT

J-GGA_B_for (29 nt)

CCAGGGTCTCTGGCTGATGTAGTTAAGGC

J-GGA_B_rev (29 nt)

GCATGGTCTCAGCCTTAACTACATCAGCC

J-GGA_C_for (29 nt)

CACCGGTCTCTGTTCTAATGGGGACCATC

J-GGA_C_rev (29 nt)

GCATGGTCTCAGATGGTCCCCATTAGAAC

J-GGA_D_for (29 nt)

AAGGGGTCTCTCGGAGCTATGTATTCCTG

J-GGA Scaffold Sequence

2013-06-11T15:42:59Z

Spolpitiyaarachchige: Created page with "J-GGA Scaffold Oligos Prefix: Gaattcgcggccgcttctagag Junction A: Atagtctgttcatggtgc Spacer: acgt Junction B: Tattgatgtagttaaggc Spacer: cgga Junction C: Tttctaatggggacca..."

J-GGA Scaffold Oligos

Prefix: Gaattcgcggccgcttctagag

Junction A: Atagtctgttcatggtgc

Spacer: acgt

Junction B: Tattgatgtagttaaggc

Spacer: cgga

Junction C: Tttctaatggggaccatc

Spacer: ttag

Junction D: Ttaagctatgtattcctg

Suffix: tactagtagcggccgctgcag

Top Strand

SCAFF_TOP1; AATTCGCGGCCGCTTCTAGAGATAGTCTGTTCATGGTGCACGTTATTGATGTAGTTAAGGCCGGATTTCTAATGG

SCAFF_TOP2; GGACCATCTTAGTTAAGCTATGTATTCCTGTACTAGTAGCGGCCGCTGCA

BOTTOM STRAND (5’ TO 3’)

SCAFF_BOT1; GCGGCCGCTACTAGTACAGGAATACATAGCTTAACTAAGATGGTCCCCATTAGAAATCCGGCCTTAACTAC

SCAFF_BOT2; ATCAATAACGTGCACCATGAACAGACTATCTCTAGAAGCGGCCGCG

Annealed AATTCGCGGCCGCTTCTAGAGATAGTCTGTTCATGGTGCACGTTATTGATGTAGTTAAGGCCGGATTTCTAATGGGGACCATCTTAGTTAAGCTATGTATTCCTGTACTAGTAGCGGCCGCTGCA

GCGCCGGCGAAGATCTCTATCAGACAAGTACCACGTGCAATAACTACATCAATTCCGGCCTAAAGATTACCCCTGGTAGAATCAATTCGATACATAAGGACATGATCATCGCCGGCG

J-GGA Scaffold Design

2013-06-11T15:41:54Z

Spolpitiyaarachchige: Created page with "J-GGA Scaffold Sequence J-GGA Scaffold Primer J-GGA Promoter Sequence Design of C dog oligos in J-GGA Scaffold Primer Design for the eCDM8 and RFP"

[[J-GGA Scaffold Sequence]]

[[J-GGA Scaffold Primer]]

[[J-GGA Promoter Sequence]]

[[Design of C dog oligos in J-GGA Scaffold]]

[[Primer Design for the eCDM8 and RFP]]

Biology

2013-06-11T15:36:52Z

Spolpitiyaarachchige:

[[Davidson Protocols]] 
[[MWSU_protocols]] 

[[J-GGA Scaffold Sequences]]

[[J-GGA Scaffold Primers]]

[[J-GGA promoter sequences]]

[[J-GGA Scaffold Design]]

[[Origin of Replication]]

[[File:Gelpouring.jpg|200px|thumb|right|Look, Mom, no EtBr!]]

'''Topics to Investigate'''

High Priority = more people

* '''Phoebe and Claire:''' eCDM8 working to make theophylline with biosensor and fitness modules (TetR) to report out. 
Which plasmid carries which device? 
Biosensor 
Fitness (tetA) 
feeds into stress module 
* '''Sachii and Jess:''' Determine the junction sequences and associated primers 
(use existing rules) 
build scaffold for insert segments 
include orI (collaborate with copy number research) 
include drug resistance gene 
test JGG design 
* '''Spencer and Sara:''' test fitness module with adhE
compare with tetA 
fine tune tetA resistance 
combine with tetA 
Does theophylline diffuse across the membrane and thus the system fails? 
* '''Brandon and Erich:''' Copy number of plasmids 
Can we alter Ori to produce a range of plasmid densities in ''E. coli''? 

Second Order Priority = one person
* produce different CDM alleles
* swap out different components (promoters, RBS, alleles)
* test out programmed evolution
* Stress module needs more work (see eCDM8 outputs)

J-GGA Scaffold Sequences

2013-06-11T15:33:34Z

Spolpitiyaarachchige:

J-GGA Scaffold Oligos

Prefix: Gaattcgcggccgcttctagag

Junction A: Atagtctgttcatggtgc

Spacer: acgt

Junction B: Tattgatgtagttaaggc

Spacer: cgga

Junction C: Tttctaatggggaccatc

Spacer: ttag

Junction D: Ttaagctatgtattcctg

Suffix: tactagtagcggccgctgcag

Top Strand

SCAFF_TOP1; AATTCGCGGCCGCTTCTAGAGATAGTCTGTTCATGGTGCACGTTATTGATGTAGTTAAGGCCGGATTTCTAATGG

SCAFF_TOP2; GGACCATCTTAGTTAAGCTATGTATTCCTGTACTAGTAGCGGCCGCTGCA

BOTTOM STRAND (5’ TO 3’)

SCAFF_BOT1; GCGGCCGCTACTAGTACAGGAATACATAGCTTAACTAAGATGGTCCCCATTAGAAATCCGGCCTTAACTAC

SCAFF_BOT2; ATCAATAACGTGCACCATGAACAGACTATCTCTAGAAGCGGCCGCG

Annealed
AATTCGCGGCCGCTTCTAGAGATAGTCTGTTCATGGTGCACGTTATTGATGTAGTTAAGGCCGGATTTCTAATGGGGACCATCTTAGTTAAGCTATGTATTCCTGTACTAGTAGCGGCCGCTGCA
GCGCCGGCGAAGATCTCTATCAGACAAGTACCACGTGCAATAACTACATCAATTCCGGCCTAAAGATTACCCCTGGTAGAATCAATTCGATACATAAGGACATGATCATCGCCGGCG

[[J]]

Biology

2013-06-11T15:32:16Z

Spolpitiyaarachchige:

[[Davidson Protocols]] 
[[MWSU_protocols]] 

[[J-GGA Scaffold Sequences]]

[[J-GGA Scaffold Primers]]

[[J-GGA promoter sequences]]

[[Origin of Replication]]

[[File:Gelpouring.jpg|200px|thumb|right|Look, Mom, no EtBr!]]

'''Topics to Investigate'''

High Priority = more people

* '''Phoebe and Claire:''' eCDM8 working to make theophylline with biosensor and fitness modules (TetR) to report out. 
Which plasmid carries which device? 
Biosensor 
Fitness (tetA) 
feeds into stress module 
* '''Sachii and Jess:''' Determine the junction sequences and associated primers 
(use existing rules) 
build scaffold for insert segments 
include orI (collaborate with copy number research) 
include drug resistance gene 
test JGG design 
* '''Spencer and Sara:''' test fitness module with adhE
compare with tetA 
fine tune tetA resistance 
combine with tetA 
Does theophylline diffuse across the membrane and thus the system fails? 
* '''Brandon and Erich:''' Copy number of plasmids 
Can we alter Ori to produce a range of plasmid densities in ''E. coli''? 

Second Order Priority = one person
* produce different CDM alleles
* swap out different components (promoters, RBS, alleles)
* test out programmed evolution
* Stress module needs more work (see eCDM8 outputs)

Biology

2013-06-11T15:31:07Z

Spolpitiyaarachchige:

[[Davidson Protocols]] 
[[MWSU_protocols]] 

[[J-GGA Scaffold Sequences]]

[[[J-GGA Scaffold Primers]]]

[[J-GGA promoter sequences]]

[[Origin of Replication]]

[[File:Gelpouring.jpg|200px|thumb|right|Look, Mom, no EtBr!]]

'''Topics to Investigate'''

High Priority = more people

* '''Phoebe and Claire:''' eCDM8 working to make theophylline with biosensor and fitness modules (TetR) to report out. 
Which plasmid carries which device? 
Biosensor 
Fitness (tetA) 
feeds into stress module 
* '''Sachii and Jess:''' Determine the junction sequences and associated primers 
(use existing rules) 
build scaffold for insert segments 
include orI (collaborate with copy number research) 
include drug resistance gene 
test JGG design 
* '''Spencer and Sara:''' test fitness module with adhE
compare with tetA 
fine tune tetA resistance 
combine with tetA 
Does theophylline diffuse across the membrane and thus the system fails? 
* '''Brandon and Erich:''' Copy number of plasmids 
Can we alter Ori to produce a range of plasmid densities in ''E. coli''? 

Second Order Priority = one person
* produce different CDM alleles
* swap out different components (promoters, RBS, alleles)
* test out programmed evolution
* Stress module needs more work (see eCDM8 outputs)

J-GGA promoter sequences

2013-06-10T04:51:02Z

Biology

2013-06-10T04:29:18Z

Spolpitiyaarachchige:

J-GGA Scaffold Primers

2013-06-10T04:26:36Z

Spolpitiyaarachchige:

J-GGA Scaffold Primers

2013-06-10T04:25:47Z

PRIMERS TO BE USED FOR INVERTED PCR OF SCAFFOLD

J-GGA_A_rev (29 nt)
GCATGGTCTCAGCACCATGAACAGACTAT
J-GGA_B_for (29 nt)
CCAGGGTCTCTGGCTGATGTAGTTAAGGC
J-GGA_B_rev (29 nt)
GCATGGTCTCAGCCTTAACTACATCAGCC
J-GGA_C_for (29 nt)
CACCGGTCTCTGTTCTAATGGGGACCATC
J-GGA_C_rev (29 nt)
GCATGGTCTCAGATGGTCCCCATTAGAAC
J-GGA_D_for (29 nt)
AAGGGGTCTCTCGGAGCTATGTATTCCTG

J-GGA Scaffold Sequences

2013-06-10T04:25:01Z

Spolpitiyaarachchige:

J-GGA Scaffold Oligos

Prefix: Gaattcgcggccgcttctagag

Junction A: Atagtctgttcatggtgc

Spacer: acgt

Junction B: Tattgatgtagttaaggc

Spacer: cgga

Junction C: Tttctaatggggaccatc

Spacer: ttag

Junction D: Ttaagctatgtattcctg

Suffix: tactagtagcggccgctgcag

Top Strand

SCAFF_TOP1; AATTCGCGGCCGCTTCTAGAGATAGTCTGTTCATGGTGCACGTTATTGATGTAGTTAAGGCCGGATTTCTAATGG

SCAFF_TOP2; GGACCATCTTAGTTAAGCTATGTATTCCTGTACTAGTAGCGGCCGCTGCA

BOTTOM STRAND (5’ TO 3’)

SCAFF_BOT1; GCGGCCGCTACTAGTACAGGAATACATAGCTTAACTAAGATGGTCCCCATTAGAAATCCGGCCTTAACTAC

SCAFF_BOT2; ATCAATAACGTGCACCATGAACAGACTATCTCTAGAAGCGGCCGCG

Annealed
AATTCGCGGCCGCTTCTAGAGATAGTCTGTTCATGGTGCACGTTATTGATGTAGTTAAGGCCGGATTTCTAATGGGGACCATCTTAGTTAAGCTATGTATTCCTGTACTAGTAGCGGCCGCTGCA
GCGCCGGCGAAGATCTCTATCAGACAAGTACCACGTGCAATAACTACATCAATTCCGGCCTAAAGATTACCCCTGGTAGAATCAATTCGATACATAAGGACATGATCATCGCCGGCG

J-GGA Scaffold Sequences

2013-06-10T04:23:53Z

Spolpitiyaarachchige:

J-GGA Scaffold Oligos

Prefix: Gaattcgcggccgcttctagag

Junction A: Atagtctgttcatggtgc

Spacer: acgt

Junction B: Tattgatgtagttaaggc

Spacer: cgga

Junction C: Tttctaatggggaccatc

Spacer: ttag

Junction D: Ttaagctatgtattcctg

Suffix: tactagtagcggccgctgcag

Top Strand

SCAFF_TOP1; AATTCGCGGCCGCTTCTAGAGATAGTCTGTTCATGGTGCACGTTATTGATGTAGTTAAGGCCGGATTTCTAATGG

SCAFF_TOP2; GGACCATCTTAGTTAAGCTATGTATTCCTGTACTAGTAGCGGCCGCTGCA

BOTTOM STRAND (5’ TO 3’)

SCAFF_BOT1; GCGGCCGCTACTAGTACAGGAATACATAGCTTAACTAAGATGGTCCCCATTAGAAATCCGGCCTTAACTAC

SCAFF_BOT2; ATCAATAACGTGCACCATGAACAGACTATCTCTAGAAGCGGCCGCG

Annealed
AATTCGCGGCCGCTTCTAGAGATAGTCTGTTCATGGTGCACGTTATTGATGTAGTTAAGGCCGGATTTCTAATGGGGACCATCTTAGTTAAGCTATGTATTCCTGTACTAGTAGCGGCCGCTGCA
GCGCCGGCGAAGATCTCTATCAGACAAGTACCACGTGCAATAACTACATCAATTCCGGCCTAAAGATTACCCCTGGTAGAATCAATTCGATACATAAGGACATGATCATCGCCGGCG

PRIMERS TO BE USED FOR INVERTED PCR OF SCAFFOLD

Biology

2013-06-09T23:40:58Z

Spolpitiyaarachchige:

[[Davidson Protocols]] 
[[MWSU_protocols]] 

[[J-GGA Scaffold Sequences]]

[[J-GGA Scaffold Primers]]

[[Origin of Replication]]

[[File:Gelpouring.jpg|200px|thumb|right|Look, Mom, no EtBr!]]

'''Topics to Investigate'''

High Priority = more people

* '''Phoebe and Claire:''' eCDM8 working to make theophylline with biosensor and fitness modules (TetR) to report out. 
Which plasmid carries which device? 
Biosensor 
Fitness (tetA) 
feeds into stress module 
* '''Sachii and Jess:''' Determine the junction sequences and associated primers 
(use existing rules) 
build scaffold for insert segments 
include orI (collaborate with copy number research) 
include drug resistance gene 
test JGG design 
* '''Spencer and Sara:''' test fitness module with adhE
compare with tetA 
fine tune tetA resistance 
combine with tetA 
Does theophylline diffuse across the membrane and thus the system fails? 
* '''Brandon and Erich:''' Copy number of plasmids 
Can we alter Ori to produce a range of plasmid densities in ''E. coli''? 

Second Order Priority = one person
* produce different CDM alleles
* swap out different components (promoters, RBS, alleles)
* test out programmed evolution
* Stress module needs more work (see eCDM8 outputs)