Part:BBa_K1390004
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1154
Illegal BamHI site found at 1188 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE 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).
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).
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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