Part:BBa_K2447014
IPTG-inducible temperature-sensitive system with GFP reporter
In the absence of IPTG, lacI repressor will bind to pLac promoter and inhibit further downstream expression. pLac promoter is attached upstream of the tlpa36 protein coding sequence to control the amount of tlpa36 protein expression. When IPTG is present and under high temperatures (above 36 degree), pTlpa36 will be constitutively and eliciting downstream GFP expression. Under low temperatures (below 36 degree), the pTlpa36 will be repressed, and drastically reducing GFP 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. 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, 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.
By replacing the weaker RBS 32 (as utilised by the Taiwanese team) with a stronger binding affinity RBS 34 as proposed by IGEM NUS team 2017, we have elucidated much stronger GFP expression and also improved the sensivity of the part to varying concentrations of phosphate ions. Previously, the Taiwanese part [http://2008.igem.org/Team:NYMU-Taipei/Project/Phosphate had reported]similar level of repressed GFP productions for phosphate concentrations above 40uM. On the other hand, our proposed phosphate sensor is sensitive to phosphate concentration above 40 uM, elucidating differential GFP expressions when placed in different phosphate level.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 1750
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1147
Illegal XhoI site found at 2035 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2338
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 3303
//collections/probiotics/control
| chassis | E.coli (beta 10) |