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System construction and optimization
To make synthesis easier and more consistent with the original strain, we decide to construct the device as shown below. We put a strong promoter J23100 upstream of constitutive AlkR, and AlkR is followed by a short terminator B1006.
Our initial device had a strong expression of RFP even without adding alkanes, which we assumed was due to 3 reasons:
a) PalkM has some leakage, to be more specific, it binds to RNA polymerase without undergoing a conformation change and activates the transcription.
b)To some extent, AlkR protein itself might form dimers in the absence of alkanes. Dimers act upon PalkM and lead to the conformation change of DNA. Then DNA binds to RNA polymerase and triggers the transcription.
c)The terminator that we choose upstream of PalkM is BBa_B1006 which is relatively short (only 34 bp), and it has a weak terminating ability, while the constitutive promoter BBa_J23100 upstream of AlkR is strong, so expression of RFP might be influenced.
We Reconstruct the gene :
After resconstructed, we transformed plasmid into our E.coli on LB plates, as the figure shows, the rate of red colony decreased to under 10%.
Results of reconstruction show that, in the absence of inducing compounds, PalkM is weak, which proves that the leakage of RFP has nothing to do with the strength of PalkM. It can also be illustrated that constitutive AlkR slightly induces PalkM, even in the absence of alkanes.
We put PalkM and AlkR on two vectors, distinguishing the causes of leakage.
And extend both sides of PalkM sequences based on the sequences reported.
The binding site of AlkR on PalkM has been reported, but it cannot be determined whether the squences on both sides of this site influence the binding of AlkR or not. For the sake of insurance, we decide to extend the length of PalkM to its upstream direction, in order to increase the sequences on both sides of the original AlkR binding site.
