Davidson Missouri W/Controlling Expression

From 2007.igem.org

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To solve the Hamiltonian Path Problem our team needs to utilize a mechanism that is capable to transcribing a sequence of adjacent genes downstream of a single promoter region. Due to the "flippable" nature of our construct, inserting a second promoter region downstream of our initial promoter region is not feasible, as we would be unable to insure that a "solved" phenotype was the result of a single path through the graph. Because of our inability to control gene expression downstream of the start of transcription, we searched for promoters of the highest processivity and repressibility. Thanks to the biobrick system we could choose from any operon in the E.Coli genome.

The Promoter Tester

We will also produce two constructs for tetsing promoters. MWSU will produce (Kan, RFP, Tet) while Davidson will produce (Kan, Tet, RFP). We can drop in different promoters and look for phenotypes.

The promoter of the Lac operon was an optimal place to start becuase the kinetics of control are well documented in comparison to most E.Coli operons.

Promoter tester.png

In addition to pLac we are going to test a lambda model promoter, the ompC porin biobrick promoter, and the T7 RNA Polymerase promoter. Davidson is also going to have synthesized an improved pLac promoter that is shorter, will have better repression, better induction, and hopefully lack the backwards promotion we have detected with Part: BBa_R0010. We will test out the modified promoter BBa_R0011 which is reported to have good repression and strong induction. We may still introduce the UV5 double mutation to enhance transcription and compare with R0010.

Davidson will also test 8 different promoters from the registry to see if any of them can promote transcription of all three genes in the promoter tester.

MWSU is also going to produce backwards LacIq to put upstream of pLac BBa_R0010. The purpose of this is to have more LacIq in the cytoplasm at all times, regardless of ITPG status.