1,721,007 research outputs found
Dataset: Bioinformatics Analysis Suggests That SE_1780 Protein From Staphylococcus Epidermidis May Be a Member of the Fph Family of Lipases
No full textStructural similarity of POI to proteins of known structure as determined by the DALI server
Model: Bioinformatics Analysis Suggests That SE_1780 Protein From Staphylococcus Epidermidis May Be a Member of the Fph Family of Lipases
No full textPyMol session file containing the overlay of PDB structures for the POI (3FLE) and B. subtilis Lipase A (1I6W)
Breaking Manufacturing Bottlenecks: A Document Control Project
Get PDFA document control project was conducted in the Manufacturing department at ***** Technologies, a San Diego biotechnology company that provides molecular diagnostics and research reagents to the hematopathology community. A 2014 quality objective for ***** Technologies dictates that a unique manufacturing batch record must be created for each cell culture processes performed by the department by the end of Quarter 1. As a result of the project detailed herein, 186 manufacturing batch records that meet current quality requirements were created and approved for the manufacturing department's cell banking and bulk cell production processes that replace the department's laboratory notebooks that have historically documented such activities. Further, the Manufacturing Work Instructions document that accompanies each batch record was updated to current department requirements. This achievement of the first part of this project represents a broken bottleneck that precluded the department from culturing the vast majority of the company's cell lines due to lack of batch records. Additionally, this achievement has increased efficiency by saving 50 labor hours for the department's supervisor from the creation and approval of affected documents. The second half of the project focused on batch records that are required that meet current quality and FDA labeling requirements for the manufacture of DNA controls included in the CE-marked in vitro diagnostic kits ***** Technologies offers to its European customers. This project achieved the created and approval of batch records for all DNA controls used in CE-marked kits. A second achievement for the second half of this project represents increased efficiency from the implementation of software that allows an author to create any amount of unique documents from a generic template. This will save the department 100 labor hours when the department creates 654 manufacturing batch records for DNA and RNA controls that ***** Technologies offers as General Purpose Reagents. This implementation will help break the second bottleneck which has prevented the manufacture of final products available for distribution or internal use due to lack of batch records
Breaking Manufacturing Bottlenecks: A Document Control Project
No full textA document control project was conducted in the Manufacturing department at *****\ud
Technologies, a San Diego biotechnology company that provides molecular\ud
diagnostics and research reagents to the hematopathology community. A 2014\ud
quality objective for ***** Technologies dictates that a unique manufacturing batch\ud
record must be created for each cell culture processes performed by the department\ud
by the end of Quarter 1. As a result of the project detailed herein, 186 manufacturing\ud
batch records that meet current quality requirements were created and approved for\ud
the manufacturing department's cell banking and bulk cell production processes that\ud
replace the department's laboratory notebooks that have historically documented\ud
such activities. Further, the Manufacturing Work Instructions document that\ud
accompanies each batch record was updated to current department requirements.\ud
This achievement of the first part of this project represents a broken bottleneck that\ud
precluded the department from culturing the vast majority of the company's cell lines\ud
due to lack of batch records. Additionally, this achievement has increased efficiency\ud
by saving 50 labor hours for the department's supervisor from the creation and\ud
approval of affected documents. The second half of the project focused on batch\ud
records that are required that meet current quality and FDA labeling requirements\ud
for the manufacture of DNA controls included in the CE-marked in vitro diagnostic\ud
kits ***** Technologies offers to its European customers. This project achieved the\ud
created and approval of batch records for all DNA controls\ud
used in CE-marked kits. A second achievement for the second half of this project\ud
represents increased efficiency from the implementation of software that allows an\ud
author to create any amount of unique documents from a generic template. This will\ud
save the department 100 labor hours when the department creates 654\ud
manufacturing batch records for DNA and RNA controls that ***** Technologies\ud
offers as General Purpose Reagents. This implementation will help break the second\ud
bottleneck which has prevented the manufacture of final products available for\ud
distribution or internal use due to lack of batch records.Biotechnolog
A Novel High Throughput Method for the Purification of Peptides Under Investigation
No full textAbstract\ud
A Novel High Throughput Method for the Purification of Peptides under Investigation\ud
Company Sponsor Mack Flinspach, PhD.\ud
Ross Fellows 04-14-2014\ud
Professional Science Masters Program, CSUSM\ud
The pharmaceutical market for peptide drugs is quickly rising, but several technical barriers to the discovery process till exist. Solid phase extraction (SPE) is a simple and crude purification method that can increase throughput and total yields of peptides under investigation and provide a competitive advantage. The standard method for peptide purification is reverse phase HPLC gradient, which regularly yields inadequate amounts for screening. Incorporating the SPE method into our process drastically increases peptide yields, increases throughput, decreases costs, and simplifies the purification. An important benefit of this method is that it can be executed with no sophisticated equipment and with minimal training. The ease of use and ability to process 96 samples in tandem allow more efficient production compared to complicated reverse phase HPLC gradients where only one sample can be run at a time. The small volume of resin used in the SPE format allows for very little organic solvent to be consumed during the process, which decreases costs and environmental waste. The total peptide yield improvement over the current method is 3-fold higher which allows molecules to be screened that would not have been possible with the previous method. The benefits of SPE come with an average of a 4% decrease in total peptide purity, which we have shown has no effect on downstream screening. Overall, the method has shown to be very efficient, reproducible, and enabled screening of peptides that would not have been possible otherwise.Biotechnolog
Effects of Zinc on Ethanol Drinking, Behavior, and Oxidative Stress Following Chronic Intermittent Ethanol Exposure
No full textThe negative effects associated with increased alcohol consumption have been well documented, and include increased risk for depression, anxiety, sleep disorders, stroke, heart disease, certain types of cancer, and permanent brain damage. It has become increasingly important to understand the genes and proteins that are associated with excessive drinking and the pathways that these genes and proteins affect. Alcohol dehydrogenase is a protein that is essential when oxidizing alcohol to acetylaldehyde using NAD as an electron acceptor. Zinc is a necessary cofactor for the alcohol dehydrogenase, without which reactive oxygen species, ROS, are created and oxidative stress damage occurs. The resulting cell damage can cause a variety of problems, including alcoholic liver disease, cardiovascular disease, and various types of cancer. The purpose of this project was to determine the effects of zinc supplementation on the amount of ethanol consumed, its effect on behavior, and oxidative stress in the brain.\ud
These were accomplished by following the chronic intermittent ethanol (CIE) model, followed by a round of behavioral testing that included a light dark transfer test, forced swim test, and conditioned fear test. Oxidative damage to the brain was determined be euthanizing the mice and removing their brains for analysis by utilizing the Mouse Oxidative Stress and Antioxidant Defense RT?? Profiler??? PCR Array available from Qiagen. A significant increase in alcohol consumption over repeated CIE cycles was found in the vapor group; however, the alcohol did not cause any significant changes in behavior nor in the amount of oxidative damage to the brain. Therefore, it is increasingly difficult to surmise that zinc may have an attenuating effect on behavioral changes and/ or a build up of oxidative damage caused by increased alcohol consumption when no such initial changes were found.Biotechnolog
Examining the Method A-Measured vs. Mathematically Calculated F/P Ratios Within the Manufacturing Procedure of FITC-Conjugated Antibodies
Get PDFXXXXXXXX continues to be a major contributor to the life science industry striving to exceed customer expectations by providing high-quality biological products. The company constantly aims to improve its manufacturing processes to strengthen its global brand. In XXXXXXXX’s XXXXXXXX site, several manufacturing employees expressed concern regarding the use of Method A within the standard operating procedure (SOP) of fluorescein isothiocyanate (FITC)-conjugated antibodies. Manufacturing operators use Method A to determine the antibody’s fluorochrome molecule:primary antibody ratio (F/P ratio). The employees claim that the Method A’s F/P ratio is drastically different from the mathematically calculated ratio. Thus, according to them, Method A is possibly causing either a weak fluorescent signal or over-fluorescence within the final products. In response to the employees’ comments and concerns, the manufacturing and operations teams launched a project to formally investigate Method A within the manufacturing SOP. A historical analysis of previous batch records indicate that Method A is overestimating the mathematically calculated F/P ratio by 19.52%. Immediately, the team sought to remove Method A from the SOP as soon as possible. However, an immediate removal of Method A from the SOP would result in a drastic problem within the manufacturing process. In the batch record analysis, the team found that only 37.4% of the antibodies’ mathematical F/P ratios were within their respective ranges. However, since Method A overestimated their F/P ratio, the antibodies were considered acceptable products to send to the customer, despite 62.6% of the antibodies not meeting their ranges. In other words, the SOP instructs the operator to only consider Method A’s F/P ratio as the numerical figure to advertise to the customer. Thus, if Method A were to be immediately removed, many of the products would have to be relabeled as the mathematical F/P ratios would be out of their ranges. Upon further investigation of the procedure’s nuances, the team discovered a variable that could significantly affect whether the final product meets its F/P ratio range. The SOP does not instruct the operator how to target an F/P ratio within the F/P ratio range. An ongoing analysis of historical batch records show that some operators aimed for the middle of the antibodies’ F/P ratio ranges, while others aimed towards the upper end of the range. In addition, the team found that the Method A and mathematical F/P ratios were significantly lower than the operators’ targeted ratio. In response, the team is currently holding pilot tests that instruct the operator to always target the maximum F/P ratio. Should the pilot tests show that the mathematical F/P ratios constantly remain within range after the adjustment, the team will make efforts to justify the removal of Method A from the SOP
Epigenetics and Exercise in Relation to Atherosclerosis
Get PDFBiologists now realize that there is more to understanding various diseases than just\ud
looking at the DNA sequence of a gene. For this reason, the study of epigenetics has\ud
grown exponentially in regards to peer reviewed articles over the last decade. The\ud
focus of epigenetics is to determine under what circumstances a gene is expressed or\ud
silenced. It is not currently understood how epigenetic factors contribute to various\ud
cardiovascular diseases, such as atherosclerosis. Atherosclerosis is the accumulation\ud
of fat and inflammatory cells in arteries. This disease was examined for the purpose\ud
of determining an epigenetic link between parents and their offspring in the study\ud
conducted. Epigenetics can be used to help understand what relationship the\ud
environment and diet has when it comes to atherogenic problems. The intent of this\ud
study was to examine whether the mother???s exercise level has any impact on their\ud
offspring; such as epigenetic changes that may be linked to atherosclerosis. This was\ud
examined by extracting RNA from the femoral artery of the infant pigs. Once RNA\ud
was extracted, cDNA was synthesized in order to perform rt-PCR. Rt-PCR was used\ud
to determine whether differential gene expression occurred between the four sample\ud
types. Qiagen RNeasy Mini Kit was determined to be the optimal protocol for\ud
extracting RNA from the femoral artery of an infant pig. Although the total amount of\ud
RNA extracted was less than the other protocols, Qiagen kit yielded the highest level\ud
of purity as well as showed clear and intact ribosomal bands when electrophoresis\ud
was performed. It could not be determined whether differential gene expression\ud
occurred between the four sample types. Efficacies of the primers used in rt- PCR\ud
were determined and a control gene (ADD1) was identified for further studies.Biotechnolog
Developing a novel PCR technology for detection of Single Nucleotide Polymorphism associated with statin Induced myalgia
Get PDFThe goal of this project was to develop a novel PCR technology for detection of single nucleotide polymorphisms (SNPs) associated with statin induced myalgia. We compared different PCR methods using sequence specific primers and UP tagged primers. Results obtained using sequence specific primers were superior to those using the same forward and reverse UP tag sequence in the primer, exhibiting higher signals and more positive analyte calls. PCR using the sequence specific primers detected five analytes not detected using the UP tagged PCR. When PCR was performed using forward and reverse UP tag primers with different UP tag sequences, the signals were improved. Seven of nine analytes detected by sequence specific PCR had strong signals with UP tagged PCR. It can be inferred from these results that the UP tagged PCR method was improved by using two different UP tag sequences in the forward and reverse primers. Further optimization of the method will be needed to apply the method to highly multiplexed PCR assays.Biotechnolog
Efficient and Multiplex genome editing using GeneArt?? CRISPR Nuclease mRNA
No full textCRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system is derived from a prokaryotic adaptive immune system that uses an RNA-guided DNA nuclease to mutate and silence viral DNA infections (Jinek et al., 2012). These nucleases have been re-engineered to function as gene editing tools in various organisms, including mammalian cells (Cong et al., 2013). Thermo Fisher Scientific has developed the GeneArt?? CRISPR Nuclease mRNA and in vitro transcribed gRNA from GeneArt?? T7 string DNA that function together to efficiently and conveniently modify the genomic loci of choice, while circumventing promoter-associated restrictions, avoiding random DNA integrations from previous DNA vector-based solutions and show potential for in vivo applications. The goal of the Biotech Master???s Semester In Residence (SIR) project is to demonstrate product and application data of GeneArt?? CRISPR Nuclease mRNA (Cas9 mRNA) system coupled with in vitro transcribed (IVT) guide RNAs (gRNAs) showing efficient editing in a broad range of cell types including human and mouse derived cell lines, as well as induced pluripotent stem cells (iPSCs). Further, to demonstrate its capabilities, we used GeneArt?? CRISPR nuclease mRNA and a set of gRNAs to simultaneously target several genomic loci at once in a multiplexed fashion and to result in clonal populations with mutations at chosen loci. Such clones were screened using the gel-based Genomic Cleavage Detection assay and confirmed through Sanger sequencing of PCR amplified targeted DNA fragments via targeted DNA via TOPO?? TA Cloning kit. The report within demonstrates how the GeneArt?? CRISPR nuclease mRNA (Catalog # A25640) can be used for performing CRISPR-Cas9 mediated genome editing. Coupled with IVT gRNA, where one can efficiently and conveniently modify their target genomic sequences. Because of eliminating promoter-associated constraints, the complete RNA format combined with RNA-specific transfection reagents, provides superior delivery combined with high genome editing efficiency in broad cell types including stem cells. GeneArt?? CRISPR Nuclease mRNA system along with the complete work flow solutions described in the report can address both small and large scale cell engineering needs.Biotechnolog
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