This project focused on optimizing the plasmid DNA purification process at Life Technologies. These plasmids are designed to code for specialized proteins used by research universities, national laboratories, or research companies. Once cultivated and harvested, the plasmids must be analyzed for quality and quantity. The project is divided into improving three aspects of the process: 1) plasmid identification, 2) plasmid purity evaluation, and 3) process yield. Plasmid identification is now simpler, more robust and has zero ambiguity. Plasmid purity evaluation is now measured with computer software, which reduces user error and eliminates subjectivity. Using the nascent metrics provided by the improved identification and purity evaluation techniques, process yield was analyzed and improved. The hypotheses on yield improvement and the information gleaned from their resulting experiments provide a foundation for further process improvement.

Non-coding DNA in the human genome is widely studied to investigate its effect on coding DNA and gene expression. Non-coding DNA contains cis-regulatory elements that influence transcription of genes upstream, downstream, or nearby. These regulatory elements have largely been studied as enhancers that promote the transcription of genes. To explore these regulatory elements as silencers, we chose validated bifunctional elements to study their silencing capability and their chromatin markers. We used chromatin immunoprecipitation methods with dCas9 to target these elements using single-guide RNAs (sgRNAs). We experimented with various cloning methods to insert dCas9 into the pUAS vector. We initially planned to use the Gibson Assembly method, but after no success, we tried site-directed mutagenesis and traditional cloning with restriction enzymes. We were able to successfully insert dCas9 into the pUAS vector with traditional cloning, and we were then able to inject the construct into Drosophila melanogaster. We designed sgRNAs to target desired elements of DNA that we chose to study as cis-regulatory elements. The sgRNA sequences were cloned into the pCFD5 vector and injected into another line of flies. The transgenic flies containing the pUAS/dCas9 plasmid will then be crossed with the flies containing the pCFD5/sgRNA to develop offspring that express the target elements and could undergo chromatin pulldown to examine the bifunctional regulation of these DNA elements in cells. Results from a quantitative PCR (qPCR) assay on Drosophila expressing the cloned pUAS vector with dCas9 and a sgRNA for the white gene showed chromatin pulldown efficiency and successful transfection. The Drosophila chromatin targeted by the sgRNAs will be pulled down, solubilized, and then analyzed on a western blot to screen for chromatin modifications, primarily histone modifications. We can then identify chromatin markers associated with elements when they act as silencers in the mesoderm versus when they act as non- mesodermal enhancers. We can also determine if the silencer acts by interacting with a promoter or with an enhancer to repress gene expression. If ENCODE can profile the data found in this project, the chromatin markers can act as a predictive tool for the identification of silencers.

The GPS2 protein (G-protein pathway suppressor 2) is a product of the mammalian gps2 gene. It was originally identified and characterized in the context of G protein mitogen-activated protein kinase (MAPK) signaling pathways. Several studies have linked GPS2 with the inhibition of the ubiquitin conjugating enzyme UBC13. GPS2-mediated inhibition of UBC13 regulates several metabolic and inflammatory pathways. It has been shown that a lack of GPS2 is correlated with an increase in adiposity and inflammation due to the aberrant activity of UBC13 affected pathways. Therefore, understanding the relationship between UBC13 and GPS2 will provide further understanding of the molecular processes involved in adipose tissue levels, inflammation and downstream molecular responses. In this study, we attempt to determine the molecular determinants of GPS2 interaction with UBC13 by optimizing the protein expression protocol required to produce GPS2 protein expression in Escherichia coli in quantities viable for biochemical and structural assays. Our results indicate that optimization of the gps2 sequence is required for efficient GPS2 protein expression in E. coli cells. Thanks to this optimization we have been able to successfully express GPS2 full length and several fragments, however, further optimization will be required for assessing GPS2-UBC13 molecular binding via in vitro binding assays.

Five strains of Lactobacillus plantarum and four strains of Lactobacillus species isolated from fresh meat were examined for the presence of plasmid DNA. All strains examined contained between one and five plasmids ranging in molecular mass from 1.3 to 51.6 (Mdal). Plasmid-curing experiments suggest that maltose utilization is associated with a 51 Mdal plasmid in Lactobacillus sp. DB29 and 42 Mdal plasmids in Lactobacillus spp. DB27, DB28, DB31. Southern blot DNA-DNA hybridization showed homology between the maltose plasmid from Lactobacillus sp. DB29 and several plasmids from the other Lactobacillus spp.

This study demonstrates the direct delivery of plasmid gene constructs into spinal motor neurons utilizing retrograde axoplasmic transport. The plasmid vectors contained the Lac Z gene under the control of both the Rous sarcoma virus (RSV) and Simian virus (SV)40 promoters. β-Galactosidase expression was observed in α and γ motor neurons by histochemical staining following direct injection into the sciatic nerve or gastrocnemius muscle. The presence of LacZ gene constructs was confirmed by the polymerase chain reaction (PCR). The ability to introduce gene constructs into motor neurons allows for the study of gene regulation and permits the development of gene therapy strategies for motor neuron diseases including the spinal muscular atrophies (SMA) and amyotrophic lateral sclerosis (ALS).

Previous small-scale methods for plasmid DNA (pDNA) purification fail to meet theindustry’s demand for sufficient quantities. Greater volumes of bacterial lysates are a consequence of larger volumetric fermentations and traditional large-scale down-stream purification processes have some disadvantages and limitations. The market is believed to continue to expand, hence the need for efficient, cost-effective, andscalable purification processes becomes apparent. A crucial trade-off exists between pDNA yield and purity, necessitating careful consideration in chromatographic pu-rification steps. Each step enhances purity while likely sacrificing yield. In order to achieve a higher degree of pDNA yield, optimal purification entails a single chro-matographic step, specifically anion-exchange chromatography (AEX) in combina-tion with filtration. Alternatively, a two-step purification approach involving AEX followed by hydrophobic interaction chromatography (HIC) is recommended to elim-inate complementary impurities and achieve a high level of purity. Furthermore, the utilization of monolithic chromatographic supports is suggested to facilitate the sug-gested purification strategies. This is due to monoliths promoting higher binding capacities, ensuring robust and consistent results even at high flow rates.

No description available.

No description available.

No description available.

Enterococci are found in soil, sewage, food, water, and are commensal to the gastrointestinal tracts of mammals, insects, and birds. Enterococci often become nosocomial pathogens that cause a wide variety of diseases including urinary tract infections, endocarditis, and septicemia. These infections are often difficult to treat with antibiotics because most of the nosocomial strains are multi-drug resistant. Enterococcal plasmids function as reservoirs for resistance genes because they are extremely stable, allow for specific and efficient transfer, and can acquire resistance determinants from the chromosome and other plasmids. Additionally, enterococcal plasmids transfer across species boundaries transferring resistance genes like vancomycin to species like Staphylococcus aureus. There are two types of enterococcal plasmids, pheromone-responsive and broad host range. Pheromone-responsive plasmids are extremely stable, have a limited host range, and are primarily found in Enterococcus faecalis. Broad host range plasmids of E. faecalis and Enterococcus faecium are less stable than pheromone-responsive plasmids, but have an expanded host range into other Gram-positive species. E. faecalis has at least 25 known pheromone-responsive conjugative plasmids. One of the most extensively studied pheromone-responsive conjugative plasmids, pCF10. Conjugation of pCF10 from donor to recipient cell is induced by pheromone cCF10. cCF10 is contained within n the lipoprotein signal sequence encoded by the E. faecalis chromosomal gene ccfA. The lipoprotein signal sequence is processed by a series of proteolytic cleavage events to produce mature cCF10. Maturation of pheromone cCF10 produces three peptides: pre-cCF10 (CcfA1-22), cCF10 (CcfA13-19), and CcfA1-12. Cells containing pCF10 continue to produce cell membrane associated precursor pheromone of cCF10 (pre-cCF10), as well as, secreted and cell wall-associated cCF10. The presence of cCF10 does not self-induce conjugation by the donor cell because of two inhibitory molecules, PrgY and iCF10. Transmembrane protein PrgY is encoded by pCF10 and reduces cell wall associated cCF10, iCF10 is a pCF10 encoded inhibitory peptide (AITLIFI) that binds to PrgX, preventing cCF10 binding. While cCF10 controls pCF10 conjugation, pre-cCF10 controls host range of pCF10 by interacting with pCF10 replication initiation protein PrgW. cCF10 can initiate conjugation and mobilize the transfer of plasmids into other species, including Lactococcus lactis, but pCF10 cannot be maintained within the cell. However, if L. lactis is engineered to produce pre-cCF10, pCF10 can be maintained. The pre-cCF10 involvement in the establishment of pCF10 into other species might be related to the observation that it binds to the pCF10 replication initiation protein PrgW. By in vitro affinity chromatography experiments, interaction of cCF10 and pre-cCF10 with PrgW induced changes in PrgW mobility in gel electrophoresis that caused by formation of doublets and formation of aggregates which were thought to be mediated by disulfide bonds. Initial evidence of regulation of PrgW conformation by disulfide bonds was seen in Western blots of E. faecalis whole cell lysates where PrgW migration is sensitive to reduction. Sequence alignment comparisons between PrgW and a group of 54 of 59 known RepA_N superfamily proteins in E. faecalis revealed three highly conserved cysteines; these RepA_N proteins had a limited host range to E. faecalis. To study the importance of theses cysteines in pCF10 maintenance and host range limitation, prgW single, double, and triple cysteine to alanine (C to A) substitutions were generated. The cysteine mutant prgW was cloned into a plasmid functioning as either a contained the prgW alone (pORI10), or containing prgW with genes necessary for efficient pCF10 maintenance (pMSP6050). While all cysteine mutant plasmids of pORI10 and pMSP6050 were still capable of replicating in E. faecalis, the plasmid stability and copy number decreased, providing evidence that the cysteines were important to PrgW function. Additionally, Western blot analysis revealed PrgW C to A substitutions decreased PrgW aggregation. Mutations of PrgW cysteines reduced pMSP6050 stability and aggregation, but increased host range to L. lactis. Both L. lactis engineered to produce pre-cCF10 and the mutation of the conserved cysteines of PrgW extended host range of pMSP6050 into L. lactis. These data taken together with the observations that pre-cCF10 induced PrgW aggregation suggested that pre-cCF10 regulated the activity of the PrgW replication initiation protein through disulfide bonds. While the conserved cysteines of RepA_N proteins are found only in E. faecalis, phylogenetic analysis revealed that RepA_N homologs lacking the three cysteines are also found in E. faecium or S. aureus, suggesting that the host range of multiple plasmids might be affected by cysteine bond formation. Phylogenetic analysis also showed that the RepA_N proteins of enterococci and staphylococci appear to have evolved to determine host range based on the presence of two of the three conserved cysteines. Modular evolution of E. faecalis plasmids, like pCF10, that contained RepA_N proteins with three conserved cysteines, might have determined the fate of the plasmid as a limited host range, stable reservoir for antibiotic resistance. / Microbiology and Immunology