Difference between revisions of "Part:BBa K2876000"

 
 
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<partinfo>BBa_K2876000 short</partinfo>
 
<partinfo>BBa_K2876000 short</partinfo>
  
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.  
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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
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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).
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[[File:P2hrep.png|400px]]
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Figure 1: Prokaryotic Two Hybrid System with pOL2-62 RFP reporter.
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[[File:RFPgraphs.png|400px]]
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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.
  
 
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===Usage and Biology===
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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.
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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).

P2hrep.png

Figure 1: Prokaryotic Two Hybrid System with pOL2-62 RFP reporter.


RFPgraphs.png

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]