Difference between revisions of "Part:BBa K2876000"

For their reporter system, Dove, Joung, and Hochschild used a strain of E. coli susceptible to knockout of histidine production in the presence of 3-AT[1]. Only when a protein-protein interaction initiates transcription of an alternate histidine production pathway would  E. coli be able to grow on his-knockout 3-AT media [1]. Because the reporter in the Dove et al system only gave a binary response of (growth if interaction) or (no growth if no interaction) we felt it was not sensitive enough for our purposes. We contacted Ann Hochschild, who was kind enough to send us the sequence for the promoter for their system, pOL2-62 (BBa_K2876000,  https://parts.igem.org/Part:BBa_K2876000), a pLacZ promoter with a modified upstream region for lambda cl binding. We then took that promoter and linked it to a turbo-RFP, to allow for a concentration dependent fluorescence response (Figure 1). To test the reporter we transformed DH5-alpha with the pOL2-62 + RFP reporter plasmid, as well as the positive control bacteriomatch lambda and alpha plasmids from Addgene (https://www.addgene.org/browse/article/8393/) and induced with IPTG. Unfortunately, when we ran the experiment on the plate reader we saw no fluorescence response (Figure 2).

For their reporter system, Dove, Joung, and Hochschild used a strain of E. coli susceptible to knockout of histidine production in the presence of 3-AT[1]. Only when a protein-protein interaction initiates transcription of an alternate histidine production pathway would  E. coli be able to grow on his-knockout 3-AT media [1]. Because the reporter in the Dove et al system only gave a binary response of (growth if interaction) or (no growth if no interaction) we felt it was not sensitive enough for our purposes. We contacted Ann Hochschild, who was kind enough to send us the sequence for the promoter for their system, pOL2-62 (BBa_K2876000,  https://parts.igem.org/Part:BBa_K2876000), a pLacZ promoter with a modified upstream region for lambda cl binding. We then took that promoter and linked it to a turbo-RFP, to allow for a concentration dependent fluorescence response (Figure 1). To test the reporter we transformed DH5-alpha with the pOL2-62 + RFP reporter plasmid, as well as the positive control bacteriomatch lambda and alpha plasmids from Addgene (https://www.addgene.org/browse/article/8393/) and induced with IPTG. Unfortunately, when we ran the experiment on the plate reader we saw no fluorescence response (Figure 2).

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