Part:BBa_K1149002

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:

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.

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.

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