Difference between revisions of "Part:BBa K2876000"

Latest revision as of 02:54, 18 October 2018

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

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 1
Illegal PstI site found at 25
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 1
Illegal PstI site found at 25
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 1
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 1
Illegal PstI site found at 25
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 1
Illegal PstI site found at 25
1000
COMPATIBLE WITH RFC[1000]

@@ Line 4: / Line 4: @@
 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).
+[[File:P2hrep.png|400px]]
+Figure 1: Prokaryotic Two Hybrid System with pOL2-62 RFP reporter.
+[[File:RFPgraphs.png|400px]]
+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.
 <!-- Add more about the biology of this part here
@@ Line 9: / Line 20: @@
 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.
-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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