Difference between revisions of "Part:BBa K2255000"

(→‎Usage and Biology)
(→‎Production of the enoyl-CoA hydratase by E. coli)
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[[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.]]
 
[[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.]]
  
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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.
+
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, 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.
+
Therefore, ''E.coli'' DH5α cells were transformed 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.  
 
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.  
Line 21: Line 21:
 
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%.
 
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.
+
Thus, 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|Result of the mass spectroscopy (MS/MSMS) analysis of the SDS-PAGE after a tryptic digestion.]]
 
[[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.]]

Revision as of 13:27, 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

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 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%.

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