Part:BBa_K1390002

mmoC

This part is an improvement of BBa_K593008. MMOC is the reductase component of the soluble methane monooxygenase from Methylococcus capsulatus (Bath) where the oxidation of NAD(P)H and the shuttling of electrons takes place [1].

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 486
  Illegal NgoMIV site found at 664
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 625
  Illegal BsaI site found at 690
  Illegal BsaI.rc site found at 733
  

Sequence

The sequence of BBa_K1390002 is identical with the actual sequence from the genome of M. capsulatus (Figure 1).

Figure 1: Alignment of BBa_K1390002 with the original sequence from M. capsulatus

Expression Analysis

To detect whether the BioBrick is synthesized in soluble form or in inclusion bodies BBa_K1390002 was provided with a 6xHis-tag, as shown for BBa_K1390008. We analyzed the expression by Western Blot. Therefore, we separated the soluble fraction and the inclusion body fraction of the subpart.

Figure 2: 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

The protein encoded in BBa_K1390002 could be expressed in the soluble fraction (Figure 2).

Summary

BBa_K1390002 contains the right DNA sequence of mmoC.
The protein encoded in BBa_K1390002 is synthesizable in the soluble fraction if expressed in E. coli JM109.

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