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Part:BBa_K1149002

Designed by: Margarita Kopniczky   Group: iGEM13_Imperial_College   (2013-09-13)
Revision as of 23:43, 2 October 2013 by Jps09 (Talk | contribs)

pelB-EstCS2-his expression (GFP reporter)

This part expresses a plastic degradation enzyme and sfGFP under the control of arabinose inducible promoter.

The enzyme is the estCS2 from BBa_K892012 previous biobrick by Washington 2012 team, that encodes carboxylesterase that breaks down ester bonds in polyurethane. We have added a pelB seretion tag to the enzyme so that it can be secreted extacellularly. We also added a 6xHistidine tag for Western blotting and protein purification purposes.

PUR degradation by esterase: PUR_esterase_theoretical_pathway_2.jpg

Characterisation

Our PUR-ESTCS2 construct contains sfGFP within an operon and therefore fluorescence can be utilised to determine if expression is being induced by addition of Arabinose.

450px-ESTCS2.png

ETSCS2 growth assay. E. coli (MG1655) transformed with ESTCS2 BBa_K1149002 were grown with either 0 μM or 6 μM Arabinose to induce ESTC2 and sfGFP expression. The growth curves appear very similar, which is confirmed by a two-tailed t-test p-value = 0.8118, thus no reason to say that there is any growth inhibition. Growth was at 37°C in LB with shaking. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

450px-ESTCS2_fluor.png ETSCS2 induction assay. E. coli (MG1655) transformed with ESTCS2 BBa_K1149002 were grown with either 0uM or 6uM Arabinose to induce ESTC2 and sfGFP expression. Induction by Arabinose produces a strong response, with induced transformants fluorescing more. Growth was at 37°C in LB with shaking. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

Conclusion: There is no growth inhibition caused by induction, but initially fluorescence from induction is elevated in PUR-ESTCS2.

References:

[http://www.microbialcellfactories.com/content/10/1/41 A novel family VII esterase with industrial potential from compost metagenomic library. Microbial Cell Factories 2011]

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
    Illegal BamHI site found at 692
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 210
    Illegal NgoMIV site found at 958
    Illegal NgoMIV site found at 1192
    Illegal AgeI site found at 2131
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1006


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Categories
//cds/biosynthesis
Parameters
None