Our favorite new Part - The climate change killer

Part: BBa_K1390019 - complete sMMO of Methylococcus capsulatus
This device consists of the six subunits of the soluble methane monooxygenase (sMMO) of Methlyococcus capsulatus (Bath). The expression is controlled by a lacI-regulated promotor BBa_R0011. The sMMO is able to convert methane to methanol using oxygen and NAD(P)H. Apart from the promoter, the BioBrick contains an RBS upstream of each subunit-encoding gene and a double terminator. The construct is therefore ready for expression in E. coli.

Our new parts

sMMO genes

We isolated all genes encoding the different subunits of the sMMO from Methylococcus capsulatus (Bath) and made them compatible with the RFC 10 Assembly Standard.

1. BBa_K1390001 - mmoB
   The mmoB gene encodes the regulatory subunit B of the sMMO
2. BBa_K1390005 - mmoY
   The mmoY gene encodes the β subunit of the hydoxylase of the sMMO.
3. BBa_K1390006 - mmoZ
   The mmoZ gene encodes the γ subunit of the hydoxylase of the sMMO.
4. BBa_K1390003 - mmoD
   The mmoD gene product might be important for the function of the sMMO, but the exact mechanism is still under investigation.

The other two sMMO genes can be found under improved parts.

Our His-tag constructs

We provided each subunit of sMMO with a 6x His-tag. This tag facilitates purification and detection of the recombinant proteins.

1. BBa_K1390007 - MMOB-His Generator
2. BBa_K1390008 - MMOC-His Generator
3. BBa_K1390009 - MMOD-His Generator
4. BBa_K1390010 - MMOX-His Generator
5. BBa_K1390011 - MMOY-His Generator
6. BBa_K1390012 - MMOZ-His Generator

Our improved parts

1. • BBa_K1390002- mmoC
     One of three subunits of the sMMO from M. capsulatus (Bath), which serves as a reductase using NADH as an electron donor.
   • Original Part: BBa_K593008 - mmo C
     The original part contains the wrong sequence without any distinct function.
   • Our improvement:
     The new part BBa_K1390002 now contains the original sequence from M. capsulatus (Bath), which was optimized for RFC10 Standard by removing restriction sites.
2. • BBa_K1390004 - mmoX
     The mmoX gene encodes the α subunit. The α subunit is one of the three proteins constituting the hydroxylase component of sMMO and houses the active site of the enzyme.
   • Original Parts: BBa_K593004 - mmo X
     The original part contained the sequence of the chromoprotein DsRedExpress instead of the mmoX sequence.
   • Our improvement:
     The new part BBa_K1390004 now contains the original sequence from M. capsulatus (Bath), which was optimized for RFC10 Standard by removing restriction sites.

For further detailed information check our Results Page and the experience pages of the original part.

Used parts

It is one of the basic principles of iGEM to build on the work of prior teams. We adapted this thought of collaborating and sharing when we built our expression constructs. In this process we used the following Standard BioBricks. We found all of these parts reliable and well-documented. These parts were provided in the iGEM 2014 DNA Distribution-Kit. For further information on why we chose exactly these BioBricks check our Results Page.

1. BBa_B0015 - double terminator
   This part was used for termination of translation and worked as expected.
2. BBa_B0032 - RBS.3 (medium) -- derivative of BBa_0030
   This part was used as a ribosome binding site. This part is known to show a relatively low strength compared to other standard RBS Bricks.
3. BBa_R0011 - Promoter (lacI regulated, lambda pL hybrid)
   This IPTG-inducible promoter was used for controlled protein biosynthesis and chosen because of the excellent documentation.
4. pSB1C3 - high copy plasmid (CmR)
   This standard plasmid was used for replication and cloning efforts.
5. pSB1A3 - high copy plasmid (AmpR)
   This standard plasmid was used for coexpression with other plasmids.

ILS

We participated in the first international interlab study, and thus helped to characterize the strength of different promoters in collaboration with teams from all over the world. In this measurement study the following BioBricks were used.

1. BBa_K823005 - strong promoter
2. BBa_K823012 - Anderson promoter J23115
3. BBa_E0240- GFP generator
4. BBa_I20260 - Measurement Kit Test of J23101
5. BBa_K516032- mRFP protein generator TERM+ RBS+(B0032)