Suppressor Logic Toolkit and Quasi-Hash Function

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Suppressor Logic Toolkit and Quasi-Hash Function

The following slides describe the basic toolkit design and a specific experiment that would result in a quasi-hash function

Basic Toolkit Design

The AND gate was designed by Anderson using termination suppressors, while the new design would use frame shift suppressors.

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Toolkit with Specific Inputs and Outputs

PprpB/propionate and luxO/pluxT or pqrr4 are new communication players that would need to be looked into further to verify their usefulness.

See references at bottom of page.

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Experimental Design

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Construct Design

NAND Gate construct

Plasmid 1

pLuxR+RBS+5 bp suppressor+TT+pPrpB+RBS+modified cI+TT approx. 1552bp

Plasmid 2

pLacI+RBS+LuxR+RBS+prpB+TT+pR+RBS+GFP+Mnt+TT+pMnt+RFP+TT approx. 4026bp

434 repressor

Design:

1. OR1 is needed. the role of OR1 is to help repressor bind to OR2 and to repress the PR;

2. OR2 is needed. The binding of the amino-terminal domain of 434 repressor to OR2 is necessary and sufficient for activation of PRM transcription;[1]

3. Need to decrease the binding affinity of OR3. binding to OR3 will cause repression of PRM transcription.Introduce a mutation in OR3 that decreases its affinity for the repressor without disturbing PRM;

4. Changing the sequence of the -35 region of PRM toward consensus greatly increases the ability of RNA polymerase to transcribe this DNA in the absence of the repressor...the mutation decreases the ability of RNA polymerase to initiate transcription at PR [2]

http://gcat.davidson.edu/GcatWiki/images/c/ce/434r.JPG

Parts Needed

Parts from Registry

Promoters

pR R00512009 Kit Plate 1 6K pSB1A2

pLacIQ1 K091112MWSU

pTetR R00402009 Kit Plate 1 6I pSB1A2

pLuxR R00622009 Kit Plate 1 6O pSB1A2

pMnt R00732009 Kit Plate 1 16B pSB2K3

Genes

RBS+lamba cI+TT P04512009 Kit Plate 2 1N pSB1AK3

pBAD+RBS+LacI+TT S03980MWSU

pBAD+RBS+tetR+TT K091145MWSU

pBAD+RBS+luxR+TT K091179MWSU

RBS(B0030)+GFP E55002007 Parts Kit Plate 1 9J pSB1A2

RBS(B0034)+RFP MWSU

Mnt C00722009 Kit Plate 1 12L pSB2K3

LuxO-P K1310222009 Kit Plate 3 8K pSB1AC3

pqrr4 K131017

Parts that need modified

LuxO-P K1310222009 Kit Plate 3 8K pSB1AC3

RBS+lamba cI+TT P04512009 Kit Plate 2 1N pSB1AK3

Parts needed to be built from scratch

prpBp Ecocyc

(prpBp text version)

tgaaattaaa catttaattt tattaaggca attgtggcac accccttgct ttgtctttat caacgcaaat aacaagttga taaca

http://gcat.davidson.edu/GcatWiki/images/2/2f/PrpBp.JPG

prpB 2-methylisocitrate lyase in E.coli MG1655 [3]http://gcat.davidson.edu/GcatWiki/images/6/6c/PrpB.JPG

(prpB text version)

atgTCTCTAC ACTCTCCAGG TAAAGCGTTT CGCGCTGCAC TGACTAAAGA AAATCCATTG

CAGATTGTTG GCACCATCAA CGCTAATCAT GCGCTGTTGG CGCAGCGTGC CGGATATCAG

GCAATTTATC TTTCTGGCGG TGGCGTGGCG GCAGGTTCGC TGGGGCTGCC CGATCTCGGT

ATTTCTACCC TTGATGATGT GCTGACCGAC ATTCGCCGTA TCACCGACGT TTGTTCGCTG

CCGCTGCTGG TGGATGCGGA TATCGGTTTT GGTTCTTCGG CCTTTAACGT GGCGCGCACC

GTGAAATCGA TGATTAAAGC CGGTGCGGCA GGATTGCATA TTGAAGATCA GGTTGGTGCG

AAACGCTGCG GTCATCGTCC GAATAAAGCG ATCGTCTCGA AAGAAGAGAT GGTGGATCGG

ATCCGCGCGG CGGTGGATGC GAAAACCGAT CCTGATTTTG TGATCATGGC GCGCACCGAT

GCTCTGGCGG TAGAGGGGCT GGATGCGGCG ATCGAGCGTG CGCAGGCCTA TGTTGAAGCG

GGTGCCGAGA TGTTGTTCCC GGAGGCGATT ACCGAACTCG CCATGTACCG CCAGTTTGCC

GATGCGGTGC AGGTGCCGAT CCTCGCCAAC ATCACCGAAT TTGGTGCCAC GCCGCTGTTT

ACCACCGACG AATTACGCAG CGCCCATGTC GCAATGGCGC TGTACCCACT TTCAGCGTTC

CGCGCCATGA ACCGCGCCGC TGAACATGTC TACAACGTCC TGCGCCAGGA AGGCACGCAG

AAAAGCGTCA TCGACACCAT GCAGACCCGC AACGAGCTGT ACGAAAGCAT CAACTACTAC

CAGTACGAAG AGAAGCTCGA CAACCTGTTT GCCCGTAGCC AGGTGAAATA A

pLuxT [4] http://gcat.davidson.edu/GcatWiki/images/e/e3/LuxT_upstream.JPG

(pLuxT text version)

agcgagcagt gagctcagtg gagtcatcag ttaaatctgt agtgttgtat tccacatttt

cgtttactgc tttcgccagc tctagatact tggcaaattc cgcttgtagc aattcttttg

aaccagcgta aatcgtcacg tctgcaacgt catcgccttc tttgatgata aagccaattt

caccagcgca accacacgct tcacatactt gggtttcttc gtgttcaatg ctcataacga

atcctcactt aaaaaaggga ttatactcaa ctcgtttttt tttcaataga aataaatgga

acaacttgga taaaaaaata tcggtatgct atgttgtttg ttgagagggt taagcaagag

ataactttag attttgttaa tggacgatgt gatccgagtc aataatttac cggatttcac

cttttactta cttagttatc acagtgagtg agttgcacct agcaagattt tataagagta

tctcgagata aattattttt acataatcta tcgctatcaa tcggttcaaa atgcagatgt

aaatattaaa ctaatgtcat ttaatggtat taatttagtt gcatattctg aaattttgtt

ttgcttgaag acatgcatct gcaaacctgg tttacagtat ttcgtaataa aaagtaggct

tgtttatttc gaaaaagggt gttttctggc aacctattga gaacgatttt cattctgacc

tcagactttg agttagatgg atttacaaag agaagagcta aaatatgcca aagcgtagta

aagaagatac cgaaatcact atccagaaaa ttatggatgc cgttgttgat cagttgctta

ggctcggtta cgacaaaatg tcgtacacga cgttgagtca acagacaggt gtttcacgta

(Xba1 site at bp81)

frame shift suppressor 1 CUGUCCUAA

frame shift suppressor 2 UUACUAGAC

prpBp and prpB have no EcoR1, Xba1, Spe1, or Pst1 sites. Source www.neb.com

References

Powerpoint File:Suppressorlogic3.ppt

Anderson et al. 2007. “Environmental signal integration by a modular AND gate” [5]

luxO luxT [6]

PprpB propionate [7]

434 repressor [8] [9]