Protein_Domain

Part:BBa_K1390004

Designed by: Stefan Dübel   Group: iGEM14_Braunschweig   (2014-07-03)

mmoX

This part is an improvement of BBa_K593004. MMOX is the α subunit of the hydroxylase component of the sMMO. It houses the active site of the enzyme where the hydroxylation of methane takes place [1].


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1154
    Illegal BamHI site found at 1188
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 163
    Illegal BsaI.rc site found at 301
    Illegal BsaI.rc site found at 718




Sequence

The sequence of BBa_K1390004 is identical with the actual sequence from the genome of M. capsulatus (Figure 1).

Figure 1: Alignment of BBa_K1390004 with the original sequence of mmoX from M. capsulatus.



Expression Analysis

To detect whether the BioBrick is synthesized in soluble form or in inclusion bodies it was provided with a 6xHis-tag, as shown for BBa_K1390010. We analyzed the expression by Western Blot. Therefore, we separated the soluble fraction and the inclusion body fraction of the BioBrick after coexpression with [http://2014.igem.org/Team:Braunschweig/Results-content#chaperone_table (chaperone plasmid C2)] from the Chaperone Plasmid Set by TaKaRa [2]. The BBa_K1390004 could be expressed in the soluble fraction with the aid of GroES and GroEL encoded on the plasmid (Figure 2).


Figure 2: Western blot of BBa_K1390010 (X) coexpressed with chaperone plasmid 2. Ladder (M): Precision Plus Proteinâ„¢ All Blue, positive control (+): His-tagged scFv, expected molecular weight: 60,6 kDa

Summary

BBa_K1390004 contains the right DNA sequence of mmoX.
The protein encoded in BBa_K1390002 is synthesizable in the soluble fraction if expressed in E. coli JM109 in combination with chaperone plasmid C2.

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
[2] Chaperone Plasmid Set Product Manual, TaKaRa Bio Inc. 2014

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