Difference between revisions of "Part:BBa K2255000"
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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. | 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. | ||
| − | [[File:T--YAU-China--iGEM2019-BBa - | + | [[File:T--YAU-China--iGEM2019-BBa -12.png|500px|thumb|none|alt=fwYellow spectum.|Figure 1. Crystal violet staining of biofilm with different volumes of extracts]] |
[[File:T--YAU-China--iGEM2019-BBa -10.png|500px|thumb|none|alt=fwYellow spectum.|Figure 2. OD570 of crystal violet of biofilm washed by 95% ethanol after adding different volumes of extract]] | [[File:T--YAU-China--iGEM2019-BBa -10.png|500px|thumb|none|alt=fwYellow spectum.|Figure 2. OD570 of crystal violet of biofilm washed by 95% ethanol after adding different volumes of extract]] | ||
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Revision as of 10:50, 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
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1093
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 16
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.
