Team:SVCE CHENNAI/TEMPERATURE SENSITIVE ACTIVATION
WHY USE TEMPERATURE SENSITIVE ACTIVATION?
In most south east Asian countries it is an indispensable practice to boil milk before consuming it. Given that we don't expect the half-life of our anti-microbial peptides to be more than a few hours , our system must be capable of producing them only when the consumer deems necessary. Hence we decided to invoke a checkpoint in the process which precedes milk consumption i.e boiling of milk.
HOW ARE WE DOING THIS?
1)Lambda pr promoter with cI mutation:
λ pR is a consituive promoter found in λ phage. It is one of the promoters that initiates the transcripion of proteins that play a role in the lytic phase of λ phage. It controls the transcripion of cell-lysisproteins, head and tail proteins. The biobrick used here contains this promoter and a mutated cI repressor protein that is the real reason forthe temperature sensitvity. Below 30⁰C, the cI repressor protein is acveand represses the acivity of the λ pR promoter. But above 35⁰C, the acivity of the repressor starts decreasing, and at 42⁰C, completelydisappears. This allows enhanced transcripon of the genes downstreamof the λ pR promoter.
This part is first characterized using ts purple chromoprotein, by measuring the expression levels of the chromoprotein at various temperatures which will be read at 500nm UV spectrophotometer.
Plasmid 1:
2)RNA thermometer:
The RNA thermometer is a temperature sensitive noncoding RNA. RNA thermometers change their secondary structure in response to temperature fluctuations. This structural change may block or expose a ribosomal binding site that may prevent or allow transcription. Mostly they are located in the 5’ UTR of mRNA encoding HSPs. The RNA thermometer used here is ROSE1 (BBa_K115001) found in Bradyrhizobiumjaponicum. As can be seen from the diagram below(courtesy: iGEM website), the shape assumed by the untranslated RNA from the RNA thermometer below 42⁰C makes transcription of any genes downstream, impossible. As the temperature increases, a hairpin structure found in the thermometer melts, allowing the transcription to occur. ROSE1 allows transcription to occur at about 42⁰C.
A plasmid construct to characterize RNA thermometer in our E.coli culture was designed with the Anderson promoter since it is well suited for protein expression in E.coli. First the RNA thermometer is ligated to the much larger cjBlue(702bp). The cjBluechromoprotein gene is followed by a double terminator.The parts activity can be studied by expression of the chromo protein at variable temperatures (30 0C – 70 0C) and OD will be measured at 610nm.
Plasmid 2:
