Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Aryo Sorayya
Date created: 2014-10-07 11:00:00
Date modified: 2015-05-08 01:10:46
quorum sensing machinery that activates GFP expression with extra terminator
| Types | DnaRegion |
| Roles | engineered_region Composite |
| Sequences | BBa_K1499503_sequence (Version 1) |
Description
This is the same part as Part:BBa_K1499500, except that it has two more terminators between the luxR gene and the luxPR promoter.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.Lux sequencing data.jpg
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| Access | Instance | Definition |
| public public public public public public public public public public public public public public public public public | BBa_R0011 BBa_B0034 BBa_C0161 BBa_B0010 BBa_B0012 BBa_R0040 BBa_B0034 BBa_C0062 BBa_B0010 BBa_B0012 BBa_B0010 BBa_B0012 BBa_R0062 BBa_B0032 BBa_E0040 BBa_B0010 BBa_B0012 | lacI+pL BBa_B0034 luxI BBa_B0010 BBa_B0012 p(tetR) BBa_B0034 luxr BBa_B0010 BBa_B0012 BBa_B0010 BBa_B0012 lux pR BBa_B0032 GFP BBa_B0010 BBa_B0012 |