Difference between revisions of "Part:BBa K2255000"

Revision as of 10:58, 16 October 2019

Enoyl-CoA hydratase

This part is the enoyl-CoA hydratase involved in the synthesis of the 2-cis-decenoic acid.

Usage and Biology

This biobrick was created to produce the enoyl-CoA hydratase, whom is an enzyme performed the formation of a double bond at the β-carbon of the decneoic acid.

Production of the enoyl-CoA hydratase by E. coli

SDS-PAGE of all the protein express in E.coli DH5α cells A) after IPTG induction and B) before IPTG induction.

The functionnality verification of this part was done by testing if E.coli was able to produced the desire enoyl-CoA hydratase and indentify by mass spectrometry if we got the right enzyme.

Therefore, E.coli DH5α cells were transformed with a pSB1C3 plasmid containing the biobrick BBa_K864400 which is a IPTG inducible promoter with a strong RBS and our BBa_K2255000, in order to produced the enoyl-CoA hydratase with an IPTG controled expression.

As you can see in the SDS PAGE, when we add IPTG in the LB-medium we observed the sur-expression of the protein (show with a black arrow) in comparaison of a native LB-medium where this massive expression is not observed. The enoyl-CoA hydratase has a molecular weight of approximatively 40 kDa, as our IPTG induced protein appeared at this weight we can assume that it correspond to our enoyl-CoA hydratase. But we need futher analysis to confirm this hypothesis.

After, the SDS-PAGE strip containing a IPTG-induced protein was cut off the gel and anlysed by mass spectroscopy (MS/MSMS) after a tryptic digestion. The mass spectroscopy analysis identify this protein as the enoyl-CoA hydratase coming from Pseudomonas aeruginosa PAO1 (NCBI database TaxID=208964). The identification was correct form the N-termini to the C-termini, with a good coverage of 86.65%.

Thus, BBa_K2255000 is a functional biobrick that will allow us production of Pseudomonas aeruginosa's enoyl-CoA hydratase.

Result of the mass spectroscopy (MS/MSMS) analysis of the SDS-PAGE after a tryptic digestion.

Sequence and Features

Characterization: YAU-China 2019

This part is an enoyl-CoA hydratase, whose main function is to participate in the synthesis of cis-2-decenoic acid which can disperse the biofilm. Therefore, our team wants to verify the damage effect of the crude cis-2-decenoic acid solution after overexpression of the enoyl-CoA hydratase gene in Pseudomonas aeruginosa on the biofilm.

We cloned the enoyl-CoA hydratase and overexpressed it in Pseudomonas aeruginosa PAO1, then extracted the crude extract of cis-2-decenoic acid, added different volumes of crude extract to culture biofilm, and observed the damage degree of biofilm.

Figure 1. Crystal violet staining of biofilm with different volumes of extracts

Figure 2. OD570 of crystal violet of biofilm washed by 95% ethanol after adding different volumes of extract

Figure 3. Destruction trend of biofilm with different volume of extract