Part:BBa_K2447501
Blue light activated inducible system with RFP reporter
In this construct, the photosensitive bacterial protein is constructively produced. In the presence of blue light, EL222 is activated via dimerisation and able to bind upstream of the luxI promoter, thereby allowing the recruitment of RNA polymerase to elicit downstream expression of RFP. In the absence of blue light, EL222 does not dimerise and unable to bind to DNA; hence eliciting minimal RFP expression.
Usage and Characterisation
This part is inserted into pBbE2k backbone and subsequently characterised in E.coli Beta 10. These cells are grown in LB medium (with kanamycin) at 30 degree for at least 48 hours before varying concentrations of IPTG ions from 0 to 500 uM are added. The cells are grown at various temperatures from 30 to 37 for 24 hours before a final GFP reading is taken.
The GFP productions correlated well to the temperature in which the cells are subjected to (figure 1). At higher temperature closer to 37 degree, there was highest amount of GFP production. On the other hand, closer to 30 degree, GFP production is repressed. This is congruent with the [http://www.nature.com/nchembio/journal/v13/n1/abs/nchembio.2233.html?foxtrotcallback=true paper's] result (figure 2) for the thermal sensitive construct which shows much higher GFP expression at 36 degree and above.
At 250uM of IPTG (figure 3), we have reported optimum condition for our temperature sensitive system. In short, our part could prove useful for any group seeking to elucidate gene expression based on temperature as a physical stimulus.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 527
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 97
Illegal AgeI site found at 1473
Illegal AgeI site found at 1585 - 1000COMPATIBLE WITH RFC[1000]
None |