Part:BBa_K2255000

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1093
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 16
  

Characterization: YAU-China 2019

We (YAU-China)verified this part. The gene contained in this part is an important gene for Pseudomonas aeruginosa to synthesize cis-2-decenoic acid, an automatic inducer of biofilm dispersion. Therefore, we carried out the following experiments: (1) We transformed the plasmid into DH5 α of Escherichia coli. (2) The crude extract of cis-2-decenoic acid was obtained by organic reagent extraction. (3) Using the crude extract of cis-2-decenoic acid to disperse the biofilm.

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Figure 1. Results of overnight treatment of biofilm with crude extract of cis-2-decenoic acid and control group

Figure 2. Quantitative ：The absorbance OD570 of the eluent of crystal violet staining of biofilm after the treatment of cis-2-decenoic acid crude extract and the control group.

Results: cis-2-decenoic acid can effectively disperse the biofilm formed by Pseudomonas aeruginosa, and there are significant differences.