The very first step of our lab work was to obtain all genes encoding the six subunits of the methane monooxygenase (mmoX, mmoY, mmoB, mmoZ, mmoD, mmoC) which catalyzes the conversion of the greenhouse gas methane to methanol, a natural intermediate of the cellular metabolism. Unfortunately, even though most of the MMO subunits are described in the iGEM Registry, most of them were not available in stock. Therefore, we isolated them directly from Methylococcus capsulatus str. Bath via PCR using appropriate self-designed primers. However, the sequence of the initial subunits mmoC, mmoX, mmoY and mmoZ contained various restriction sites within the genes (mostly PstI). In order to receive the RFC10 standard these were removed by using mutation PCR. We then cloned them into the pSB1C3 shipping vector to put them at disposal of the community of all future iGEM teams.
For detailed information, take a look at our Lab notebook E. 1 Isolation of sMMO genes from M. capsulatus.

Now that we had successfully isolated the genes for all the MMO subunits, our next goal was to proof that we were able to produce all of them in the well characterized and easily manageable model organism Escherichia coli. In order to check for expression later on, we added a His-tag at the C-terminus of each subunit by using appropriate PCR-primers. Afterwards, we put them under control of the inducible lac-promoter (R0011), a weak ribosome binding site (B0032) and a double terminator (B0015).
For detailed information, take a look at our Lab notebook E. 2 HIS-Tagging of mmo genes.

Expression of His-construct

Plasmids containing the final His-tagged constructs were then transformed into chemo-competent E. coli cells and the single tagged subunits were expressed by induction with IPTG. To isolate the soluble and inclusion protein fractions, the cells were harvested and pelleted by centrifugation. SDS-PAGE followed by an immunostain via His-tag was performed in order to verify the expression of the subunits. Unfortunately, only two subunits were detectable (MMOC and MMOX). For the purpose of producing all of the MMO subunits efficiently we tested the coexpression of different combinations of known chaperone proteins. With their help the E. coli cells were able to express each of the six MMO subunits in soluble form. Hence, we are the first to efficiently produce all of these MMO subunits in the heterologous organism E.coli.
For detailed information, take a look at our Lab notebook E. 5 Expression of HIS-constructs.

After ensuring that all the subunits of the MMO were producible in E. coli using the right combination of chaperones, we decided to assemble all of them in one construct, thus creating our methane degrading E. cowli! In order to allow a subsequent induction of our final construct containing all six of the MMO subunit genes we again used the lac-promotor in combination with a weak RBS and a double terminator. In order to make sure that every subunit-encoding gene is translated, a single RBS is inserted upstream of every gene.

In order to allow a subsequent induction of our final construct containing all six of the MMO subunit genes we again used the lac-promotor in combination with a weak RBS and a double terminator. In order to make sure that every subunit-encoding gene is translated, a single RBS is inserted upstream of every gene.
For detailed information, take a look at our Lab notebook E. 3 Cloning of final construct.

Expression of the mmo construct

To analyse the functionality of the final construct we cultivated our E.cowli in a converted anareobic jar filled with a 2% methane atmosphere for 6 h. In order to proof the activity of the enzyme complex we measured the decrease of the methane concentration over a time period of 3 h with an MQ-4 semiconductor sensor for natural gas, a kind gift from iGEM Team Aachen.
For detailed information, take a look at our Lab notebook E. 6 Expression of mmo construct.

For our coexpression experiments we used different combination of chaperones. Therefore, we created a stock of competent E. coli cells, that were made chemically competent after transformation with the different chaperone constructs.
For detailed information, take a look at our Lab notebook E.7 Preparing of competent cells with chaperones.

In order to test our constructs under realistic conditions, we performed various rumen fluid experiments. Fresh rumen fluid was kindly provided by the Institute of Animal Nutrition, Friedrich-Loeffler-Institute (FLI), Federal Research Institute for Animal Health, Braunschweig and steril filtrated. We then used this steril fluid to analyse our E.Cowli’s chances in the cow’s rumen.
For detailed information, take a look at our Lab notebook E. 7 Rumen fluid experiments

Based on our first rumen fluid experiments, we have learnt that E. coli is not capable to grow on rumen fluid. In order to improve the chances of survivial of our E. cowli, as well as, to find a media to transport our E. cowli into thr cow's rumen we tested the impact of entrapment of E. cowli in alginate beads and analysed its growth.
For detailed information, take a look at our Lab notebook E. 8 Entrapping of E. coli.

In this year’s iGEM competition the first international Interlab Measurement Study was introduced to obtain fluorescence data for three specific genetic devices expressing GFP. Even though we did not sign up for the track “Measurement” of the iGEM competition and therefore were not obliged to participate in this Interlab Measurement Study, we voluntarily took part as one of 45 teams worldwide. In addition to the general requirements we also completed the second “extra credit” assignment by building and testing all three devices expressing RFP instead of GFP and compared the fluorescence of both fluorescence proteins.
For detailed information, take a look at our Lab notebook Interlab Study.