Filtering by
- All Subjects: E. coli
- Creators: Chemical Engineering Program
- Creators: Varman, Arul
Stable glycerol stocks of the pIbpA-antiPBAD and pIbpA-antiHtdR in BW25113 cells with either a pBLN200 or pHtdR200 plasmid were created. Then after inducing the cells with L-arabinose and 10mM all-trans retinal to allow for membrane protein expression, spectrophotometry was used to test the optical density of the cells at an absorbance of 600nm. Although general trends showed that the pHtdR200-pMM102 and pHtdR200-pIbpA cells had lower optical densities than the pBLN200 cells of all types, the results were determined to be statistically insignificant. Continuing, the pHtdR200 cells of all types showed a purple phenotype when spun down, as expected, while the cells with the pBLN200 plasmid had a colorless phenotype in pellet form. Further work will include cloning a GFP gene-block to test the ability of the anti-PBAD sequence in tuning the transcription of the GFP protein.
The increased shift towards environmentalism has brought notable attention to a universal excessive plastic consumption and subsequent plastic overload in landfills. Among these plastics, polyethylene terephthalate, more commonly known as PET, constitutes a large percentage of the waste that ends up in landfills. Material and chemical/thermal methods for recycling are both costly, and inefficient, which necessitates a more sustainable and cheaper alternative. The current study aims at fulfilling that role through genetic engineering of Bacillus subtilis with integration of genes from LCC, Ideonella sakaiensis, and Bacillus subtilis. The plasmid construction was done through restriction cloning. A recombinant plasmid for the expression of LCC was constructed, and transformed into Escherichia coli. Future experiments for this study should include redesigning of primers, with possible combination of signal peptides with genes during construct design, and more advanced assays for effective outcomes.
allow E. coli to consume xylose and glucose, two ubiquitous and industrially relevant microbial feedstocks, simultaneously was implemented and systematically evaluated for its effects on L-phenylalanine (Phe; a precursor to many microbially-derived aromatics such as 2PE) production. Ultimately, by incorporating this mutation into a Phe overproducing strain of E. coli, improvements in overall Phe titers, yields and sugar consumption in glucose-xylose mixed feeds could be obtained. While upstream efforts to improve precursor availability are necessary to ultimately reach economically-viable production, the effect of end-product toxicity on production metrics for many aromatics is severe. By utilizing a transcriptional profiling technique (i.e., RNA sequencing), key insights into the mechanisms behind styrene-induced toxicity in E. coli and the cellular response systems that are activated to maintain cell viability were obtained. By investigating variances in the transcriptional response between styrene-producing cells and cells where styrene was added exogenously, better understanding on how mechanisms such as the phage shock, heat-shock and membrane-altering responses react in different scenarios. Ultimately, these efforts to diversify the collection of microbially-produced aromatics, improve intracellular precursor pools and further the understanding of cellular response to toxic aromatic compounds, give insight into methods for improved future metabolic engineering endeavors.