Results

General Anoxic HGT Biosensor Bioreactor Design

Anoxic Results

Preparing pAWP78 for Gene Insertion

To linearize pAWP78 before subsequent insertion of the desired genes, a polymerase chain reaction (PCR) was carried out on the plasmid followed by a DPNI digestion. The expected size of pAWP78 is shown in the agarose gel (Figure 2). Issues arose with amplification of pAWP78, however successful amplification was achieved with the inclusion of 9% Dimethyl Sulfoxide (DMSO) and higher temperatures of the gradient 60-80 ^oC. After amplification, a plasmid extraction was conducted, yielding a 117.45 ng/uL yield.

**Figure 2a (LEFT) and 2b (RIGHT):** 2a) Gel Electrophoresis of Polymerase Chain Reaction of backbone pAWP78 with a DMSO gradient, using a 1kb+ ladder as a marker. The 7 wells on the bottom row represent PCR of pAWP78 with a 9% concentration of DMSO. 2b) to verify a 9% concentration worked, another gel electrophoresis was performed. The band in the third well is the desired size, at approximately 5.3 kb.

Gibson Assembly, Transformation of Constructs and Plasmid Purification; Summer 2019

With the synthesized inserts and the purified, linearized backbone, the entire construct can be assembled utilizing Gibson assembly. The initial Gibson assembly was completed using a 2:1 ratio of pAWP78 backbone to insert. From the colonies that grew, a colony PCR was performed, but yielded the wrong size band, as we were expecting a band around 3.5 kB. A transformation was again performed with the same Gibson assembly, however, commercially electrocompetent DH5 alpha E. coli cells were used from NEB. When checking for growth, none was observed. Following this, various Gibson assemblies were created using different ratios, different concentrations, and different competent cells.

Gibson Assembly, Transformation and Purification; Fall 2019

Before the process of assembling the plasmid could be repeated, various PCRs were conducted on each insert and on pAWP78 to amplify them for a higher concentration. The two genes for construct B, DAMO_2415 and DAMO_2437 were gel extracted along with the amplified backbone pAWP78. A Gibson assembly was created using a 1:1 and 3:1 insert to backbone ratio, transformed via heat shock into DH5 alpha cells and subsequently plated on LB + Kanamycin plates. No growth was observed. The Gibson assembly was repeated with adjustments in volumes for the 1:1 and 5:1 assemblies. There were 8 colonies present on the 1:1 Gibson assembly and 1 present on the 5:1 plate, along with 3 present on the negative control. The negative control that was used was a 40 uL sample of DH5 alpha competent cells without a plasmid, while the positive control was the plasmid pHC61 with a kanamycin resistance marker. Significant growth was present on the spot plates for each colony. A colony PCR yielded amplification at a slightly smaller size than expected, as the band was at approximately 1.5 kB when the desired size was approximately 3.5 kB. The potential candidates were sent out for sequencing, but no priming occurred. Because of this, if the failure to prime error was not caused by an excessive concentration of the primers, we can conclude that the colony is likely not the plasmid with the inserted genes. For future work, the goal would be to assemble and transform the desired genes into 20Z. Following this, a growth assay should be run to test if the genes work as expected.

**Figure 3** Gel electrophoresis of potential candidate DH5 alpha cells containing plasmid. Purified DNA from colony 1 is in lane 3, purified DNA from colony 2 is in lane 5 and the negative control empty plasmid is in lane 7. From this gel, it can be concluded that the two purified colonies do not contain the empty plasmid, as the band size appears to be different from that of the empty plasmid. However, the expected size of the plasmid with the two inserted genes is not present, so it can not be concluded that this is the plasmid with both genes.

Growth Assay

A growth assay was conducted on wild type Methylomicrobium alcaliphilum 20Z, in which the strain was grown in several different conditions. The strain was grown using methanol and methane as a carbon source, and under varying oxygen levels including fully oxic, microaerophilic, and fully anoxic.

The results for the growth curve aligned with what is known about the M. alcaliphilum 20Z strain. Under conditions without oxygen, growth was stunted in both the methanol and methane trials. In contrast, M. alcaliphilum 20Z continued growing in oxic and microaerophilic conditions. A growth curve for all six conditions was constructed (Figure 4.) From these results, the understanding that M. alcaliphilum cannot function under anoxic conditions at the same rate as with oxygen, and it can also be concluded that methanol acts as a better carbon source than methane. In the future, should this plasmid be able to be transformed into M. alcaliphilum, one would have the ability to compare growth rates under each condition to verify that the inserted genes were able to help the bacteria function under anoxic conditions.

**Figure 4.** Methanol Growth Curves with 20Z

**Figure 5.** Methane Growth Curves with 20Z