Part:BBa_K2876000
placOL2-62 E. coli promoter
This is an E. coli lacZ promoter with a modified upstream lambda-cl bonding site used for transcription in a E. coli two hybrid system. The sequence for this promoter was donated by Ann Hochschild
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 took this 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).
Figure 1: Prokaryotic Two Hybrid System with pOL2-62 RFP reporter.
Figure 2: 48hr plate reader fluorescence experiment of pOL2-62 + RFP reporter with bacteriomatch system (positive control lambda and alpha fusion proteins) and induced with IPTG.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1
Illegal PstI site found at 25 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1
Illegal PstI site found at 25 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1
Illegal PstI site found at 25 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1
Illegal PstI site found at 25 - 1000COMPATIBLE WITH RFC[1000]
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