Introduction
Anaerobic digestion of dairy farm waste products such as wastewater and biosolids into useful energy-rich has been implemented in large farms throughout the world. However, the biogas derived from such systems, although have been shown to reduce methane emission, are not easily collected due to inefficient gas to liquid conversion within the system. Researchers have proposed the addition of methanotrophic bacteria as a possible solution due to their ability to utilize methane as a carbon energy source. However, maintenance of the methanotrophic bacteria are difficult and not cost-effective. Therefore, by taking inspiration from the 2014 Braunschweig and 2021 Wageningen teams, we wished to engineer an E. coli strain capable of expressing the soluble methane monooxygenase enzyme (sMMO) from Methylococcus capsulatus. sMMO can catalyze the conversion of methane into methanol, which can then be readily extracted as energy source.
Construct design
The biobrick design of the 2014 Braunschweig team inspired us to adapt their design. The biobrick (BBa_K1390019) designed by the team included all 6 genes that encodes the different subunits of the soluble MMO. The construct was placed into pSB1A3 and transformed into E. coli JM109. In addition, the Braunschweig team co-transformed two chaperones GroEL and GroES using Takara Clontech’s Chaperone Plasmid C2. (see below for schematic of the biobrick design).
In our design, we decided to create several modifications that we believe can increase the expression and therefore activity level of the soluble sMMO:
- Codon optimization. In the original design, the mmo genes were obtained from the genome of M. capsulatus. We decided to optimize all 6 ORFs to codons suitable for E. coli expression. With this design, we created BBa_K4884006 (see below).
- Promotor optimization. In the second design, we decided to swap out the promoter with the inducible AraBAD promoter. We believe this stronger promoter might also enhance expression. Furthermore, since the chaperone carrying plasmid from Takara Biotech also uses an arabinose inducible promoter, this design would allow us to induce both the sMMO subunits as well as the chaperones. With this design, we created BBa_K4884006 (see below).
Results:
Due to time constraints and also funding issues, we decided to submit the two biobrick constructs to IDT for full synthesis. Since we codon optimized all the mmo genes, this was really the only way. In addition, we decided to request IDT to clone the entire construct into their pUC57 based plasmids which encodes for ampicillin resistance. We were successful in obtaining the plasmids from IDT.
We next transformed the two plasmids into E. coli DH5alpha and selected on Amp 100 LB agar plates. After obtaining single colonies, we made glycerol stocks. We also were successful in transform the Takara chaperone Plasmid C2 into these two E. coli strains and selected for both plasmids on LB agar containing ampicillin and chloramphenicol.
Unfortunately, due to time constraints we were unable to test the expression of the sMMO enzyme in our system.
Development of a rapid fluorescent based assay to detect sMMO activity
A critical assay for any team that works with sMMO would be to confirm the activity of the sMMO enzyme. Namely, whether it has successfully converted methane into methanol. The 2014 Braunschweig team created an anaerobic jar in which methane concentration was detected via a MQ-4 semiconductor sensor. In our literature search, we identified another potentially cheaper alternative. In a 2002 publication by MIller et al, they discovered that coumarin can be oxidized in the presence of a functional sMMO into 7-hydroxycoumarin. The emission spectrum of 7-hydroxycoumarin is significantly different from that of coumarin to be detected and quantified using a fluorometer.
Results:
Based on the protocol described by Miller et al, we were able to purchase coumarin and obtained Methylosinus trichosporium cell lysates. Unfortunately, the only fluorometer that was accessible to us at OSU broke when we attempted to use it, and we were unable to find another alternative.
Summary and future directions:
- We successfully obtained our new sMMO expressing plasmid and transformed them into E. coli DH5alpha along with plasmid C2 from Takara Biotech carrying E. coli GroEL and GroES.
- We ran out of time to test our E. coli strains for expression of the subunits.
- We devised a different way of measuring sMMO activity based on the oxidation of coumarin ino a fluorogenic substrate 7-hydroxycoumarin. All raw materials were successfully obtained, unfortunately due to equipment failure we were not able to set up the assay.
- Being the first ever iGEM team developed at OSU, we worked hard in forming a talented team of students and we were able to obtain funding so we could enter the competition. We wished we had more time and better luck in testing our E. coli strains. However, this process is now ongoing in our team.
- Once we have confirmed the activity of sMMO in our engineered E. coli, we would like to go a step further in building a prototype of our own fluorometer to create a mockup of what we envision our engineered bacteria can do in real life applications.
- We would also like to mutate the amino acid residues of the sMMO subunits in an attempt to increase enzyme activity. A result publication by Bennett et al (2021) performed directed evolution and identified many mutations that resulted in higher activity. We would like to repeat some of the experiments to see if it works in our case.
References:
https://2014.igem.org/Team:Braunschweig
https://2021.igem.org/Team:Wageningen_UR
Miller, A., et al. "A rapid fluorescence-based assay for detecting soluble methane monooxygenase." Applied microbiology and biotechnology 58 (2002): 183-188.
Bennett, R. Kyle, et al. "Expression of soluble methane monooxygenase in Escherichia coli enables methane conversion." BioRXIV (2021): 2021-08.