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.
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 +
====Production of the enoyl-CoA hydratase by '''E. coli'''====
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 +
[[File:T--Aix-Marseille--P0744-EP.png|300px|right|thumb|]]
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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 [https://parts.igem.org/Part:BBa_K864400 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.
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 +
[[File:T--Aix-Marseille--P0744-MS.png|800px|centre|thumb|]]
  
 
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Revision as of 13:18, 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

T--Aix-Marseille--P0744-EP.png

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.

T--Aix-Marseille--P0744-MS.png

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