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Team:TUDelft/Temperature design
From 2008.igem.org
>> work in progress
Parts Design
First approach three excisting RNA thermometers and one artificial RNA thermometer based on G-quadruplex.
Scar problem
Turning the RNA thermometer sequences into standard biobricks is not as straightforward as adding a prefix and suffix to the found sequences. The reason for this is that the start codon of the protein coding part following the RNA thermometer is also part of the RNA thermometer sequence (figure ... sequences met atg overlap). The problem with this approach is that the ligation of the two parts results in a sequence containing two start codons with in between the scar (figure ... wrong situation sticking together two parts). The first of these start codon (the one on the RNA thermometer) will act as the initiation point for the translation, because of its distance to the Shine Dalgarno sequence. At first some extra amino acids will be added to the protein, but even worse, the protein will not be translated at all. This is because of the scar that contains a stop codon. So the translation will already end before the protein coding region is reached.
To solve this problem we have to alter the sequence of the RNA thermometer. Alteration of the protein coding part is of course no option because we are aiming for a standard part that can be combined with any other standardized protein coding part. So these parts, that always start with a start codon should be kept intact. The simple solution is to chop of the last part of the RNA thermometer sequence, which will be replaced by the scar and the start of the protein coding part after ligation (figure ... solution without scar adaption).
But this results in a new problem. The alteration of the RNA thermometer sequence also changes its secondary structure and that will also affect its function as temperature sensor (figure ... change of secondary structure). To resolve this we made some extra alteration in the RNA thermometer sequence at the opposite position of the scar in order to regain the original secondary structure.
