Team:MichiganState/Design/Anoxic

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Project Design

General Anoxic HGT Biosensor Bioreactor Design

Methylobacterium species are gram-negative bacteria that are found readily in places such as soil, sewage and leaf surfaces. They meticulously utilize methanol, or another carbon source like C2, C3, and C4.¹ Methanotrophs, a type of methylotroph, oxidize methane in the presence of oxygen and uses methane as the carbon source for metabolism. Because methane is a product of anaerobic digestion, methanotrophs are abundant in areas where this digestion takes place like wetlands, rice paddies, mud pots, and alkaline-rich lakes among others.

In deep portions of landfills where organic matter is degraded anaerobically, methane is released and dissipates into our warming atmosphere. This particular environmental issue prompted us to use methanotrophs, or methane metabolizing bacteria, as our means to a solution. Methanotrophs only work in the presence of oxygen, therefore it was necessary to find a way to modify the bacterium to work under the anoxic conditions of the landfill. A particular methanotrophic bacterium strain, Candidatus Methylomirabilis oxyfera (NC10)5, has the ability to create its oxygen in the NC10 Pathway5³. Nitrite reductase (nir) converts Nitrite to Nitric oxide and Nitric oxide dismutase (nod) likely converts Nitric oxide to Nitrogen gas and oxygen gas. The idea of NC10 Pathway5 creating its oxygen was vital.

Figure 1 Predicted pathway containing nir and nod genes

This pathway is unique for methanotrophs, therefore it is advantageous to implement it into another strain: Methylomicrobium alcaliphilum 20Z4. This strain is ideal for landfill applications because, while methanotrophs typically output carbon dioxide, strain 20Z has a pre-existing genetically engineered pathway that allows for a sustainable solution. This existing engineered pathway is attributed to Marina G. Kalyuzhnaya from San Diego State. The methane metabolism pathway outputs 2,3-Butanediol, a compound that can be converted into biofuel ².

Implementation of the genes from the NC10 strain into the 20Z strain was the basis for this environmental project. The nir gene is well characterized for converting Nitrite to Nitric oxide ³. However, the nod gene has not explicitly been proven to convert Nitric oxide to Nitrogen gas and oxygen gas. It is only suspected that is the function, therefore, there are two nod paralogs, DAMO_2434, and DAMO_2437 which need to be used to discern which explicitly converts Nitric oxide to Nitrogen gas and oxygen gas.

Three constructs are created to test the two nod paralogs in conjugation with the verified nir gene. Construct A is comprised of the nir gene with the DAMO_2434 nod gene. Construct B is comprised of the nir gene with the DAMO_2437 nod gene. Construct C is comprised of the nir gene in conjugation with both nod paralogs to see if the function is based on both paralogs.

Figure 1a (LEFT), 1b (CENTER) and 1c (RIGHT): 1a) The proposed modified plasmid known as plasmid A, including promoter pmoA, nitrate reductase (nir, DAMO_2415) and a variation of the nitric oxide dismutase nod (DAMO_2434). Primers are included for reference on the plasmid pAWP78 to see where intended priming was to occur. In this case, burnt orange represents pAWP78, neon green represents pmoA, blue represents DAMO_2415 and tan represents DAMO_2434. 1b) The proposed modified plasmid known as plasmid B, including pmoA, DAMO_2415 and a variation of nitric oxide dismutase, nod, DAMO_2437. The colors correspond to the same genes as in a with the exception that tan now represents DAMO_2437. 1c) The proposed modified plasmid known as plasmid C, including promoter pmoA, DAMO_2415, DAMO_2434 and DAMO_2437. In this case, the colors remained the same with the addition of a new color: DAMO_2437 is represented by light orange in this model, while DAMO_2434 is represented by tan.

These inserts are to Gibson assembled with the pAWP78 plasmid and to be transformed into the 20Z strain directly. The sequencing and phenotypic assay testing come with the successful transformation of the 3 constructs into the 20Z strain. A baseline growth curve of the 20Z strain in anoxic, microaerophilic, and oxic conditions is necessary to then compare to the 20Z containing the plasmid. The growth assay will allow testing of the phenotype of the genes inserted by the plasmid.

References

¹ Aken, B. V. (2004). Methylobacterium populi sp. nov., a novel aerobic, pink-pigmented, facultatively methylotrophic, methane-utilizing bacterium isolated from poplar trees (Populus deltoidesxnigra DN34). International Journal Of Systematic And Evolutionary Microbiology, 54(4), 1191–1196. doi: 10.1099/ijs.0.02796-0

² DucNguyena, A., G.Kalyuzhnaya, M., YeolLeea, E., & AbstractMethane. (2018, April 16). Systematic metabolic engineering of Methylomicrobium alcaliphilum 20Z for 2,3-butanediol production from methane.

³ Ettwig, K. F.; Speth, D. R.; Reimann, J.; Wu, M. L.; Jetten, M. S. M.; Keltjens, J. T. Bacterial Oxygen Production in the Dark. Frontiers in Microbiology 2012, 3.