Part:BBa_K1499503:Design

quorum sensing machinery that activates GFP expression with extra terminator

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1952
  Illegal BsaI.rc site found at 2680
  

Design Notes

After ligating the BBa_K1499500 construct together and into the BioBrick backbone, we transformed it into the NEB 5-alpha strain of E. coli. The colonies that fluoresced were most likely to have been successfully transformed, and thus these were sent off for sequencing. The sequencing data showed that our construct was correct (see below), so we were able to submit for BioBricking.

Although our sequencing data was good, we realized that we had not induced the lac-repressible promoter, and thus the colonies should not have been fluorescing yet. After doing more research, we realized that the strain of E. coli we were using was not lactose deficient, thus the quorum sensing cascade was being activated by the natural presence of lactose. We ordered a lacI^q strain of E. coli, which are lac deficient, so that we could have complete control of GFP expression. However, when we performed the transformation of the construct into this strain, all of the colonies still fluoresced.

After doing further research, we discovered that the luxPR promoter (BBa_R0062) [2], will induce backward transcription if the luxR protein is present but the AHL molecule is not. In the lac deficient strain of E. coli, AHL is not produced without IPTG induction, but luxR expression is controlled by the constitutive ptet promoter. We hypothesize that since luxR was present in these cells without AHL, the backward transcription from luxPR hindered the function of the terminators that separated the luxR gene from the GFP gene. Thus GFP may have been expressed because the ptet promoter, which is constitutive, was able to affect its expression.

In order to bypass this issue, inserted two more terminators between the luxR gene and the luxPR promoter, in hopes that this would stop the backward transcription from having an effect on GFP expression, allowing us to work towards measuring the time delay created by our construct. The double-promoter did help reduce the GFP expression, though not completely.

Source

BBa_K1499500