Part:BBa_K1390008

MMOC-His Generator

This is the His-tagged version of mmoC from Methylococcus capsulatus under control of Lac-promotor R0011 with RBS B0032 and the terminator B0032.

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 568
  Illegal NgoMIV site found at 746
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 707
  Illegal BsaI site found at 772
  Illegal BsaI.rc site found at 815
  

Biology and Usage

MMOC is the reductase component of the soluble methane monooxygenase where the oxidation of NAD(P)H and the shuttling of electrons takes place [1].

Design and Coexpression

We put BBa_K1390008 under the control of the inducible lacl promoter (R0011) which has a low leakiness and is easily inducible with IPTG. This strong, frequently used promotor is well characterised in the iGEM registry and is reported to be well functioning. We also added the extensively documented weak ribosome binding site (B0032) with a strength of 33.96% compared to B0034 to diminish the formation of inclusion bodies and a double terminator (B0015). For expression we decided to use pSB1A3 because of its ampicillin resistance (in contrast to the chaperone plasmid which carries a chloramphenicol resistance cassette).

Expression Analysis

To detect whether the subunit is synthesized in soluble form or in inclusion bodies it was provided with a 6xHis-tag. We analyzed the expression by Western Blot. We separated the soluble fraction and the inclusion body fraction of the subpart. The subpart could be expressed in the soluble fraction (Figure 1).

Figure 1: Western blot of BBa_K1390008 (C) in the soluble (sol) and inclusion body (IB) fractionsLadder (M): Precision Plus Protein™ All Blue, expected molecular weight: 38,6 kDa

Summary

BBa_K1390008 is synthesizable in the soluble fraction.

Reference

[1] Wang W, Iacob RE, Luoh RP, Engen JR, Lippard SJ (2014) Electron Transfer Control in Soluble Methane Monooxygenase. J Am Chem Soc 136:9754-62