Part:BBa_K1055007:Experience
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Applications of BBa_K1055007
This part is an improvement of the last years BioBrick BBa_K808025 to control and observe its expression even better then before.
To achieve this goal, first, the FSC coding sequence has to be amplified out of the pET22b(+):BBa K808025 plasmid (derived from Sven Jager, TU-Darmstadt). This plasmid includes the FSC gene fused to the gene of mRFP1 (Part:BBa_E1010). Due to this, we were able to quantify the production process of the FSC. Moreover, this approach will result in a convenient screening of mutants and wild type enzyme, using IPTG in LB agar plates (e.g 0.1 to 0.5mM). The amplified gene will be in frame with the pelb leader sequence in order to direct the FSC to E.colis' periplasm. This feature is important for the maturation of the three disulfide bonds. These bonds are only formed under oxidative conditions. Without this feature the FSC will be unfunctional. Additionally, we take the ribosom binding site from the pET22b(+) vector. Moreover, to achieve high purity of the protein, the FSC fusion gene will get a His-Tag at the C-terminus. Therefore, we designed overlap primers with the corresponding motive. This feature is important for the purification of the protein of interest. For an easy cleavage of the reporter as well as the His-Tag, we found a TEV site between the N-Terminus of the FSC and the C-terminal region. Furthermore, to optimize the expression and thus receiving a higher protein yield, the FSC (in frame with the pELB leader sequence) will be cloned in front of an arabinose inducible promotor (e.g. pBAD (PID: BBa I0500)). For the backbone we used the overlap fragment from the TLO-CMK inFusion approach.
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