Part:BBa_K1718006
Genetic Switch LuxI GFP
This part consists of a switch composed of a strong Anderson promoter and a terminator flanked by attB and attP, the recognition sites for BxbI. In its off position, the terminator prevents expression of the downstream part. If the BxbI invertase is expressed, it will recognize the attB and attP sites and invert the DNA; thus, flipping the terminator so polymerase can pass through the gate. The Bxb1 invertase flips DNA using the attB and attP sites.
After the switch the are sequences that code for luxI, the autoinducer synthetase for AHL, and GFP.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 55
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 962
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 64
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 84
Illegal BsaI.rc site found at 245
Illegal BsaI.rc site found at 1638
Characterization of Switch with LuxI-GFP
Preliminary results with RFP and GFP showed that the switch was leaky when on a pSB1C3 backbone. We believe that because of the high copy number of pSB1C3, there was a high number of switches in each cell so if only a small proportion of switches were to leak, this effect would be visible. In order to investigate this issue, we transfered the gate with a GFP reporter onto a low copy plasmid. As expected, we saw much less expression of our reporter.
To test the responsiveness of our integrase in tandem with the leakiness of the gate on a low copy plasmid we co-transformed the Bxb1 integrase behind the arabinose inducible promoter on a kanamycinbackbone with BBa_K1718005 (switch-LuxI-GFP) on 4C5.
It was tested by growing cells in overnight cultures with varying concentrations of arabinose, from 0% to .2%increasing by .04% for a total of 6 cultures. The cultures were made of 5mL of LB, 5ul of both chloramphenicol and kanamycin, and cells taken from a glycerol stock of cells with the desired plasmids. The 4th culture had low growth.
Flow cytometry was performed on the cells after being resuspended in PBS which returned the resulting data.
With the exception of the fourth sample, which was the culture with low growth, we see a general rightward trend in the peaks, indicating that higher concentrations of arabinose are more effective at inducing the production of the integrase. More tests should be done to confirm this trend and find the ideal concentration.
| None |