PETase can catalyze the hydrolysis of PET plastic to monomeric mono-2-hydroxyethyl terephthalate (MHET). MHET is then further broken down by MHETase to terephthalic acid and ethylene glycol.
Our gblocks are composed of BioBrick Prefix and Suffix, which have EcoRI and PstI cut sites. We perform restriction enzymes cut using these two cut sites for ligation to pET-Blue2, which is a cloning and expression vector. The plasmid will be transformed into NEB 5-alpha strain Escherichia coli for cloning, as well as BL21(DE3) strain E. coli for T7 expression. Nevertheless, we found that the GC content of both enzymes are high, which caused the failure of PCR. We then try to optimize the codon. As a result, we successfully complete the PCR, ligation and transformation.
Moreover, carbon dioxide produced in the process is a greenhouse gas. Using it as a carbon source for the process of carbon reduction to methane can further utilize the product of PET decomposition, and also benefits our environment.
Carbon dioxide Reduction to Methane using Modified Nitrogenase
It has been suggested that through site directed mutagenesis of nifD, which encodes the α-subunit of nitrogenase, CO2 is converted to CH4 instead of N2 to NH3 through electron reduction. In previous study, double site directed mutagenesis was conducted in order to introduce V75A and H201Q in nifD of. Rhodopseudomonas palustris. This altered the substrate binding site of FeMo nitrogenase., conferring the ability of CO2 reduction.
Further studying the amino acid substitution in nitrogenase, it shows that through site directed mutagenesis, we can control the binding of different substrate and restrict the access of N2 gas in the atmosphere. Then, bound substrate can be converted to another substance.
Using the minimal gene set, consists of only 9 nif gene, we try to express the amino acid substituted nitrogenase in E. coli BL21 strain using its native promoter.
Through Gibson assembly, we assemble the five DNA fragments consist of the 9 nif genes with the native promoter found in Paenibacillus Polymyxa WLY.78. To find the correct construct, we use the pETBlue-2 for Blue White Screening before the construct is transformed to pSB1C3.
In our project, we propose a simple yet spectacular MoFe nitrogenase induced CO2 to CH4 in E. coli. Here, CO2, commonly known to be a harmful gas to the environment which contributes to Global Warming among the society, can be converted useful substance, i.e. CH4. CH4 is known to have the ability to generate electricity. Given that it is a renewable energy source, there is a high potential that method mentioned is feasible.