Projects

Downstream Effects of pgi gene Knockout and Manipulation of Media on E. Coli and Bacteriophage Replications

Many illnesses are caused by viral infections but they are poorly understood on how they affect the metabolic pathways, especially in Escherichia coli, for successful replication. Thus, we investigated a gene called pgi that codes for glucose-6-phosphate isomerase, an enzyme used in the second major step of glycolysis when glucose is oxidized into fructose. We examined the effects of T2, T4, and T4r phage replication by removing pgi in E. coli and predicted that the removal will decrease bacteria growth and phage replication.

To explore the role of the pgi gene in phage replication, we used a parent strain of E. coli and another with the pgi removed. We also manipulated LB and M9+glucose media, to observe different environmental effects on bacteria growth and phage replication. We performed plaque assays, two-time point experiments, and utilized the plate reader to obtain growth and lysis curves in two trials to characterize the strains and phage replication.

Our results demonstrated that plaques formed with the knockout were smaller than that of the parent. Also, fewer plaques were formed with T4 and T4r phages. Interestingly, the knockout strain grew faster in both LB and M9+glucose, contradicting our prediction. From the lysis curves, T4 is the best phage for lysis as the absorbances decreased overtime and T4r with pgi revealed a battle between the phage and the bacteria. However, the starting absorbances were not similar and the controls were lysed in M9+glucose, therefore, no conclusion. Lastly, from the two-time points, pgi allowed greater growth of T2 yet inconsistent while the parent allowed steady increase growth. However, the strains’ growth was slower in M9+glucose and the control for M9+glucose was contaminated. For future directions, we will repeat the lysis curve and the two-time points in M9+glucose media for better results.

The Role of Aspartate Metabolism On E. Coli and Bacteriophage Replication

Bacteriophage are viruses that hijack bacteria, such as E. coli, in order to manipulate the bacterial host’s metabolic pathway to successfully replicate. Research has demonstrated that the absence of aspC, which encodes a multifunctional enzyme called aspartate aminotransferase, negatively affects E. coli growth. Utilizing this information, we investigated the impact of the absence of aspC on bacteriophage replication due to its role in aspartate metabolism.

Our experimental approach began with characterizing growth of wild type and ΔaspC E.coli in various minimal media formulations supplemented with glucose, glutamine, and aspartate by utilizing a plate reader to continuously monitor growth through spectroscopy. Our results demonstrated that ΔaspC had deficits in replication compared to wild type in LB media, M9 glucose, M9 glucose/glutamine, and M9 glucose/glutamine/aspartate. This confirms that aspC possesses a significant role in E. coli metabolic pathways. Additionally, supplementation of glutamine and aspartate increased the rate of ΔaspC growth, which supports research that aspC catalyzes a reaction that produces L-glutamate with L-aspartate.

Following the growth curves, we observed wild type and ΔaspC E.coli lysis when exposed to T-even (T2, T4, T4r) bacteriophage in the plate reader to continuously monitor growth through spectroscopy. Results demonstrated that T2 bacteriophage was not able to efficiently lyse the wild type or ΔaspC, but the T2 bacteriophage replicated less in ΔaspC overall. T4 bacteriophage was able to lyse ΔaspC more effectively but at a similar rate when compared to the wild type. T4r bacteriophage demonstrated significant deficits in replication in ΔaspC, which may suggest that rapid lysis may be prohibited by the absence of aspC.

Additionally, we investigated T-even bacteriophage in the wild type and ΔaspC E.coli by quantifying at multiple time-points. Quantifying T2 and T4r bacteriophage at multiple time-points did not provide reasonable results that could be analyzed due to unprecedented errors. Future work will aim to further characterize E. coli and bacteriophage replication when exposed to aspartate by utilizing the plate reader and two time-point phage titer experiments.

The Effect of E. Coli THRA on Bacteriophage Replication

In recent years, there has been an increase in antibiotic-resistant bacteria related deaths world-wide. With antibiotics becoming less effective, it’s crucial that we find an alternative treatment option. Phage therapy has emerged as a novel treatment for bacterial infections and has even shown to work in a few recent cases. Unfortunately, however, the host-pathogen interaction between the bacterial cell (host) and bacteriophage virus (pathogen) is poorly understood.

Our focus was to further understand this relationship through the manipulation of the host cell (Escherichia coli) through using genetic knockouts to determine the impact of viral replication. It is already known that amino acids are essential for building proteins, which in turn are essential in both cell replication and cell metabolism. However, genes that encode for these amino acids can be removed and then replaced with a selectable marker, thus blocking the synthesis of such amino acids. The cell will still be capable of replicating, however, the effect it would have on bacteriophage replication is poorly studied.

We assessed the effect of bacteriophage replication (T2 and T4r) with the knockout of the thrA gene in the host cell. This gene is one of the key pathways in the synthesis of L-homoserine - an essential amino acid. We first characterized the growth of the knockout strain, and then characterized T2 and T4r phage replication in the ΔthrA host cell. Our knockout strain has shown to have a similar growth curve as the parent strain (BW25113) in LB media, while there was no growth in M9 glucose media. We also saw a decrease in T4r phage replication with the ΔthrA as the host cell, however, T2 phage replication has greatly increased in the knockout strain. Future work will aim to rescue growth in the knockout strain through supplementing M9 glucose media with amino acids.

RESEARCH POSTERS