Latest revision as of 15:56, 17 October 2014

MMOY-His Generator

This is the His-tagged version of mmoY 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
INCOMPATIBLE WITH RFC[12]
Illegal NotI site found at 315
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 272
Illegal NgoMIV site found at 1108
Illegal AgeI site found at 519
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 338

Biology and Usage

MMOY encodes the β subunit of the hydroxylase component of the sMMO [1].

Design and coexpression

We put BBa_K1390011 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). To ensure proper folding of the BBa_K1390011 in E. coli JM109 we coexpressed it with chaperones (groES, groEL). The TaKaRa Clontech Chaperone Plasmid Set proved to be appropriate for this as it contains a chaperone plasmid encoding groES and groEL ([http://2014.igem.org/Team:Braunschweig/Results-content#chaperone_table chaperone plasmid C2]) which can be coexpressed with BBa_K1390011 [2]. Synthesis of the protein can be induced as soon as the chaperones are operational.

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 after coexpression with chaperone plasmid 2. The subpart could be expressed in the soluble fraction with the aid of GroES and GroEL encoded on the plasmid (Figure 1).

Figure 1: Western blot of BBa_K1390011(Y) coexpressed with chaperone plasmid 2. Ladder (M): Precision Plus Protein™ All Blue, positive control (+): His-tagged scFv, expected molecular weight: 44,70 kDa

Summary

BBa_K1390011 is synthesizable in the soluble fraction by use of several chaperones. We recommend the use of GroEL/ GroES provided by Takara (Takara Bio Inc., Japan).

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

@@ Line 25: / Line 25: @@
 =Expression Analysis=
-To detect whether the subunit is synthesized in soluble form or in inclusion bodies it was provided with a 6xHis-tag, as      shown for BBa_K1390011.
+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 after coexpression with chaperone plasmid 2. The subpart could be expressed in the soluble fraction with the aid of GroES and GroEL encoded on the plasmid (Figure 1).

Difference between revisions of "Part:BBa K1390011"