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Agrobacterium tumefaciens has the ability to transfer its tumor inducing (Ti) plasmid into plant cells. In the last decade, agroinfiltration of Nicotiana benthamiana plants has shown promising results for recombinant protein production. However, A. tumefaciens produce endotoxins in the form of lipopolysaccharides (LPS), a component of their outer membrane that can induce organ failure and septic shock. Therefore, we aimed to detoxify A. tumefaciens by modifying their Lipid A structure, the toxic region of LPS, via mutating the genes for lipid A biosynthesis. Two mutant strains of A. tumefaciens were infiltrated into N. benthamiana stems to test for tumor formation to ensure that the detoxifying process did not compromise the ability of gene transfer. Our results demonstrated that A. tumefaciens with both single and double mutations retained the ability to form tumors. Thus, these mutants can be utilized to generate engineered A. tumefaciens strains for the production of plant-based pharmaceuticals with low endotoxicity.
ContributorsHaseefa, Fathima (Author) / Chen, Qiang (Thesis director) / Mason, Hugh (Committee member) / Hurtado, Jonathan (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Protein and gene circuit level synthetic bioengineering can require years to develop a single target. Phage assisted continuous evolution (PACE) is a powerful new tool for rapidly engineering new genes and proteins, but the method requires an automated cell culture system, making it inaccessible to non industrial research programs. Complex protein functions, like specific binding, require similarly dynamic PACE selection that can be alternatively induced or suppressed, with heat labile chemicals like tetracycline. Selection conditions must be controlled continuously over days, with adjustments made every few minutes. To make PACE experiments accessible to the broader community, we designed dedicated cell culture hardware and integrated optogenetically controlled plasmids. The low cost and open source platform allows a user to conduct PACE with continuous monitoring and precise control of evolution using light.
ContributorsTse, Ashley (Author) / Bartelle, Benjamin (Thesis director) / Tian, Xiaojun (Committee member) / Barrett, The Honors College (Contributor) / Materials Science and Engineering Program (Contributor) / School of International Letters and Cultures (Contributor) / Harrington Bioengineering Program (Contributor)
Created2023-05
Description
Abiotic stresses, such as heat, can drive protein misfolding and aggregation, leading to inhibition of cellular function and ultimately cell death. Unexpectedly, a thermotolerant Escherichia coli was identified from a pool of antibiotic resistant RNA polymerase β subunit (rpoB) mutants. This stress tolerant phenotype was characterized through exposure to high temperature and ethanol. After 30-minute exposure of cells to 55°C or 25% ethanol, the mutant displayed 100 times greater viability than the wild-type, indicating that the rpoB mutation may have broadly affected the cellular environment to reduce protein misfolding and/or prevent protein aggregation. To further test this hypothesis, we examined thermotolerance of cells lacking heat shock chaperone DnaJ (Hsp40), which is a cochaperone of one of the most abundant and conserved chaperones, DnaK (Hsp70). The deletion of dnaJ led to severe growth defects in the wild-type, namely a slower growth rate and extreme filamentation at 42°C. The severity of the growth defects increased after additionally deleting DnaJ analog, CbpA. However, these defects were significantly ameliorated by the rpoB mutation. Finally, the rpoB mutant was found to be minimally affected by the simultaneous depletion of DnaK and DnaJ compared to the wild-type, which failed to form single colonies at 37°C and 42°C. Based on these observations, it is proposed that the rpoB mutant’s robust thermotolerant phenotype results from a cellular environment protective against protein aggregation or improper folding. The folding environment of the rpoB mutants should be further examined to elucidate the mechanism by which both antibiotic resistance and thermotolerance can be conferred.
ContributorsYeh, Melody (Author) / Misra, Rajeev (Thesis director) / Wang, Xuan (Committee member) / Kelly, Keilen (Committee member) / School of Life Sciences (Contributor) / School of International Letters and Cultures (Contributor) / School of Human Evolution & Social Change (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05