Difference between revisions of "Part:BBa K2255000"
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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. | 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 | + | ====Production of the enoyl-CoA hydratase by ''E. coli''==== |
| − | [[File:T--Aix-Marseille--P0744-EP.png|300px|right|thumb|]] | + | [[File:T--Aix-Marseille--P0744-EP.png|300px|right|thumb|SDS-PAGE of all the protein express in ''E.coli'' DH5α cells A) after IPTG induction and B) before IPTG induction.]] |
In order to verify the functionnality of this part, we choose to test if ''E.coli'' was able to produce the desire enoyl-CoA hydratase and indify by mass spectrometry if we have the right enzyme. | In order to verify the functionnality of this part, we choose to test if ''E.coli'' was able to produce the desire enoyl-CoA hydratase and indify by mass spectrometry if we have the right enzyme. | ||
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Therefore, the BBa_K2255000 is a functional biobrick that will allow us production of ''Pseudomonas aeruginosa'''s enoyl-CoA hydratase. | Therefore, the BBa_K2255000 is a functional biobrick that will allow us production of ''Pseudomonas aeruginosa'''s enoyl-CoA hydratase. | ||
| − | [[File:T--Aix-Marseille--P0744-MS.png|800px|centre|thumb|]] | + | [[File:T--Aix-Marseille--P0744-MS.png|800px|centre|thumb|Result of the mass spectroscopy (MS/MSMS) analysis of the SDS-PAGE after a tryptic digestion.]] |
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Revision as of 13:21, 25 October 2017
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
In order to verify the functionnality of this part, we choose to test if E.coli was able to produce the desire enoyl-CoA hydratase and indify by mass spectrometry if we have the right enzyme.
Therefore, we transformed E.coli DH5α cells with a pSB1C3 plasmid containing the biobrick BBa_K864400 and 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 (circle in black) in comparaison of a native LB-medium where this massive expression is not observed.
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%.
Therefore, the 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