Team:TU Darmstadt/labbook/Cutinase

In order to improve a part of last year, we choose a functional production construct with the excellent PET degrading enzyme: Fusarium Solani Cutinase.
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.

Method

For the construction we used a three part assembly with the excellent inFusion method . First, we have to apply an overlap extension PCR to all parts of the final construct. After purifying all parts, we performed the inFusion reaction. Parts for the inFusion reaction: pSB1C3 (100ng), pBAD(30ng) and FsC-mRFP1(150ng). Fortunately, we could use the overlapped backbone from the inFusion approach from TLO-CMK.

Primers

• >pET|OEPCR|FOR
  • CGG CCG CTA CTA GTA TCA GTG GTG GTG GTG GTG GTG A (37 bp)

• >pET|OEPCR|REV
  • GGC TAG CAA GGA GAT ATA CAT ATG AAA TAC CTG CTG CC (38 bp)

• >pBad|OEPCR|FOR
  • CGG CCG CTT CTA GAG ACA TTG ATT ATT TGC ACG GCG T (37 bp)

• >pBad|OEPCR|REV
  • ATC TCC TTG CTA GCC CAA AAA AAC GGT ATG GAG (33 bp)

Results

First, the cloning approach, using inFusion yields in numerous positive clones. Afterwards, the clones were screened with the standard primers vr and vf. The inFusion reaction yields in 4 positive clones.

Lanes 1 and 2 of the agarose gel show very slight DNA bands at ca. 1500 bp. Lanes 5 to 9 show very distinct bands at < 300 bp.

The gel shows the result of a colony pcr against the cutinase construct FSC-mRFP1. The lanes 1,2 and 6-9 show DNA bands of ca. 2000 bp, indicating positive clones.

The final vector construct contains three major parts: the pelB-FsC-mRFP-6xHis construct, the pBAD promotor and the pSB1C3 backbone.