Difference between revisions of "Part:BBa K2255000"

(→‎Production of the enoyl-CoA hydratase by E. coli)
(→‎Characterization: YAU-China 2019)
 
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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.
 
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.
  
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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.
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Therefore, ''E.coli'' DH5α cells were transformed with a pSB1C3 plasmid containing the biobrick [https://parts.igem.org/Part:BBa_K864400 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.
  
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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 39 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.
+
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%.
 
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%.
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<partinfo>BBa_K2255000 parameters</partinfo>
 
<partinfo>BBa_K2255000 parameters</partinfo>
 
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==Characterization: YAU-China 2019==
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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 <i>Pseudomonas aeruginosa</i> on the biofilm.
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 +
<br>
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We cloned the enoyl-CoA hydratase andabundantly expressed in <i>Pseudomonas aeruginosa</i> lacking the DspI gene, using an empty plasmid as a control, and then extracting the crude extract of cis-2-decenoic acid, added different volumes of crude extract to culture biofilm, and observed the damage degree of biofilm.
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[[File:T--YAU-China--iGEM2019-BBa -12.jpg|500px|thumb|none|alt=Crystal violet staining of biofilm with different volumes of extracts.|Figure 1. Crystal violet staining of biofilm with different volumes of extracts]]
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[[File:T--YAU-China--iGEM2019-BBa -10.png|500px|thumb|none|alt=Destruction trend of biofilm with different volume of extract.|Figure 2. Destruction trend of biofilm with different volume of extract]]
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  Legend: Black is 40 μl of empty plasmid extract, gray is added with different volumes of cis-2-decenoic acid crude extract
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[[File:T--YAU-China--iGEM2019-BBa -11.png|500px|thumb|none|alt=Destruction trend of biofilm with different volume of extract.|Figure 3. Destruction trend of biofilm with different volume of extract]]
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Results: Cis-2-decenoic acid can destroy the biofilm effectively. With the increase of the volume of the extract, the destruction degree of the biofilm is greater. At 30-40 μL, the damage degree reaches the maximum value.
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<br>

Latest revision as of 01:05, 22 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

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

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 andabundantly expressed in Pseudomonas aeruginosa lacking the DspI gene, using an empty plasmid as a control, and then extracting the crude extract of cis-2-decenoic acid, added different volumes of crude extract to culture biofilm, and observed the damage degree of biofilm.

Crystal violet staining of biofilm with different volumes of extracts.
Figure 1. Crystal violet staining of biofilm with different volumes of extracts
Destruction trend of biofilm with different volume of extract.
Figure 2. Destruction trend of biofilm with different volume of extract
 Legend: Black is 40 μl of empty plasmid extract, gray is added with different volumes of cis-2-decenoic acid crude extract
Destruction trend of biofilm with different volume of extract.
Figure 3. Destruction trend of biofilm with different volume of extract

Results: Cis-2-decenoic acid can destroy the biofilm effectively. With the increase of the volume of the extract, the destruction degree of the biofilm is greater. At 30-40 μL, the damage degree reaches the maximum value.