Difference between revisions of "Team:UMaryland/Description"

Background

When solar energy is absorbed by the Earth’s surface, the energy radiates back and is then absorbed by greenhouse gases. This absorption causes heat to stay at the Earth’s surface, creating a warming effect. Methane is a potent greenhouse gas in the atmosphere that, though not as abundant as other greenhouse gases, has a major affect on the warming of the planet. The three biggest contributors to methane emissions are fossil fuel production, livestock farming, and landfills.

Different ways of managing methane emissions and reducing the amount of methane production are being to put to use. Legislation passed by the EPA requires Oil and Gas companies to monitor their methane emissions and are required to operate under regulations that require emissions to be under a certain quantity. Methane in landfills is managed by flaring or used in power generators that function as an alternative energy source.

Livestock produce methane gas in their metabolism and manure produces methane as it decomposes. In attempt to control the amount of methane produced by the latter, farms can implement anaerobic digesters that use the biogases produced by anaerobic digestion and decomposition of manure to create an alternative energy or heat source.

However, regulations do not stop the problem, flaring is not 100% effective and can produce air pollutants, and machines that are able to use methane as an energy or heat source can be expensive and difficult to maintain.

A biological alternative to reducing methane in the atmosphere is the implementation of methanotrophs, prokaryotes that metabolize and use methane as their main carbon source.

Design

As a way to sequester methane emissions, we will engineer E. coli to be able to metabolize methane. To do this, we have designed two metabolic pathways that take methane to biomass or carbon dioxide.

• Both pathways start with the conversion of methane to methanol using the sMMO gene found in Methylococcus capsulatus, a methanotroph. This gene produces an enzyme that can oxidize methane, therefore converting it to methanol.
• Once the methane is converted to methanol, other engineered E. coli will metabolize the methanol. This will be able to happen in two different pathways that we have designed.
• The first pathway, as we call the fructose pathway, uses a series of enzymes to convert the methanol to a fructose derivative.
• The second, or alternative pathway, as we call the formate pathway, will convert the methanol to carbon dioxide, a much less potent gas, and NADH, a molecule commonly used in redox reactions.
• The engineered bacteria will be co-cultured to provide the complete pathway of methane metabolism.
• This co-culture can be applied to the piping of landfills, where methane gas may escape.

Results

sMMO

The first plasmid, termed “sMMO” , is adopted from Team Braunschweig 2014. This plasmid has the sMMO gene that produces the soluble form of methane monooxygenase, which converts methane to methanol.

We attempted to recreate this plasmid using several different methods. The plasmid consists of a lac pL promoter, inducible by IPTG, six sMMO subunits, a ribosome binding site before each subunit, and a double terminator. The subunits are MMOB, MMOC, MMOD, MMOX, MMOY, and MMOZ. MMOB, MMOC, MMOD, and MMOX were all special ordered from the registry. The lac pL promoter and ribosome binding site were obtained from the kit. An RBS, MMOY, another RBS, MMOZ, and the double terminator were designed into a G-Block provided by IDT.

We designed the G-Block to have an overhang on both ends that corresponded to the ends of a linearized backbone and then performed a gibson assembly to insert that set of genes into a chloramphenicol resistant backbone provided by iGEM. We then attempted to combine a ribosome binding site with each subunit using 3A assembly. The plan was then to sequentially combine the subunits together, combine them with the G-Block part, and then combine the promoter with that using more 3A assemblies. Unfortunately, we were not able to find success in this strategy and the length of the process proved to be a major setback so we looked to try a new method.

For this new method, we hoped to take advantage of the ease of gibson assemblies. We designed primers for each subunit. The forward primer would contain an overhang to correspond to the part it would attach to as well as a ribosome binding site. The reverse primer would simply anneal to the end of the subunit, excluding the final subunit, which would also contain an overhang that would correspond to the backbone. The products of the PCR reactions would then be put into a gibson reaction with a plasmid that had a chloramphenicol resistant backbone and an insert of the lac pL promoter. The proposed order would be MMOB, MMOC, MMOD, and MMOX, with MMOB having an overhang with the end of the lac pL promoter, MMOC having an overhang with MMOB, MMOD having an overhang with MMOC, and MMOX having an overhang with MMOD (in the forward primer) and the backbone (in the reverse primer). This method, too, was not providing us the results we were expecting.

Our final attempt at creating the sMMO plasmid involved purchasing two new G-Blocks, one with the promoter, a ribosome binding site, and MMOX, and another with ribosome binding sites in front of MMOB, MMOC, and MMOD. These G-Blocks were designed with overhangs to be combined with each other and be used in a gibson reaction with a chloramphenicol resistant backbone to create a plasmid. This plasmid was then to be combined with the MMOY/MMOZ plasmid using a 3A assembly.

The next plasmid we assembled was our “Fructose” plasmid. This plasmid, adopted from Team Aachen 2015, directs a metabolic pathway that converts methanol to formaldehyde with the MDH2 enzyme (methane dehydrogenase), then converts formaldehyde to 3-hexulose-6-phosphate with the HPS enzyme (3-hexulose-6-phosphate synthase), and converts that product to D-fructose 6-phosphate using the PHI enzyme (6-phospho-3-hexuloisomerase). This enzymatic pathway converts methanol to a fructose derivative.

To assemble this part we ordered two G-Blocks, one containing a lac pL promoter, a ribosome binding site, and MDH2, and another containing ribosome binding sites in front of HPS and PHI and a double terminator. The G-Blocks were designed so that they had overhangs that corresponded with each other as well as the ends of the backbone. We performed a gibson assembly to put all the pieces together and got our final product of the assembled plasmid.

To test the functionality of our plasmid we transformed BL21 with the construct. We performed an SDS PAGE to analyze if the protein was being expressed. We also measured the methanol concentration over time when the cells were grown in methanol setting under induction.

Our final plasmid, the “Formate” plasmid, was adopted from Team UESTC-China 2014. We designed a plasmid with the same MDH2 gene as the Fructose plasmid, an FDH gene, and an FALDH gene. The pathway created by these enzymes converts methanol to carbon dioxide and NADH by first using MDH2 to convert methanol to formaldehyde, using FALDH to convert formaldehyde to formate, and using FDH to convert formate to the final product. To assemble this plasmid we attempted to