Dna microarrays also will be utilized to examine the effect of hsp overexpression on global gene expression. Identification and Study of Other Genes That Might be involved in Solvent Tolerance. We will manufacture larger dna arrays (to examine the expression profile of 1,100 genes; this will cover about 25 percent of the. Acetobutylicum genome) to examine the effects of solvent challenges on the expression of a large number of genes. Genes that show large differences in differential expression will be cloned and characterized, and their overexpression will be examined for the ability to confer increased solvent tolerance using an approach identical to the one described above for the hsps. Journal Articles: no journal articles submitted with this report: view all 23 publications for this project Supplemental keywords: dna arrays, transcriptome., rfa, scientific Discipline, waste, sustainable Industry/Business, genetics, Environmental Chemistry, sustainable Environment, health Risk Assessment, technology for Sustainable Environment, biochemistry, bioremediation, bioprocessing, gene expression patterns.
Progress / Interim Reports
The arrays also contain several positive and negative controls. The expression profile of both butanol- and heat-stressed cells was determined. Cells were exposed to butanol (1percent) and heat (42?C) for 10 minutes. The data show that transcription levels change after only 10 minutes of stress. Exposure to butanol alters the expression of genes that have been implicated in the stress response (chaperonins) and of other genes that usually are not implicated in stress pathways. Some genes involved in butanol synthesis were upregulated, while other solvent-production related genes had relative low transcript levels. Future Activities: The following future activites will be performed: overexpression of Dnakj and Groesl. Plasmid constructs for overexpression of Dnakj and Groesl genes will be methylated and transformed into. A new strategy, outlined above, will be used to successfully methylate and transform Clostridia with these constructs. These strains will be characterized by fermentation and flux analysis as mba well as by northern and Western analysis.
Thl promoter) has been methylated and inserted into. A new strategy is now undertaken to make methylation of these plasmids possible. Rather than transforming the desired plasmid into. Coli in which pAN1 is already established,. Coli carrying the desired plasmid will be made electrocompetent and then transformed with pAN1. Expression Analysis of the butanol-Stress Response Using dna arrays a dna-microarray protocol has been developed for expression analysis. We constructed dna arrays containing 96 to 500 genes, all pcr amplified from. Genes have been selected from the following classes/pathways: glycolysis and sugar metabolism, solvent and acid production, the heat-shock response, antibiotic resistance, sporulation, business and energy generation.
Coli clone in which the Groesl and Dnakj genes were successfully inserted into the vector did not result in a positive clone. It has been shown that father's overproduction of hsps is toxic in a number of hosts (including. We hypothesized that transformation and growth. Coli at a lower temperature (25oC) may decrease the initial level of stress created by increased hsp production. This hypothesis proved to be correct in so far as it resulted in the identification of positive clones: positive clones grew up to 1,000 times better at 25?c versus cells grown at 37?c, as determined by cfu/mL counts. Acetobutylicum atcc 824 can be transformed with these four constructs, it is necessary to methylate the constructs by first passing them through the methylating strain. Plasmid pAN1 contains the f3t i methyltransferase gene that methylates Cac824I restriction sites. Similar temperature-related difficulties have been encountered with this transformation. One of the four constructs (pgroe1?
A second probe to the dnaK/dnaJ genes confirmed this finding. A third probe to the Groesl operon resulted in.2kb band corresponding to the expected transcript size. The.2kb band was an order of magnitude more intense on the wild type blot relative to the orf a overexpressing strain. This is the first direct evidence that orf a acts as a repressor of the Dnakj and Groesl operons. Overexpression of Dnakj and Groesl operon Genes we cloned a fragment of the Dnakj operon containing all three functional genes (grpE, dnak, and dnaJ). Similarly, the Groesl operon genes (groes and groEL) were amplified without its natural promoter or the circe element. The amplified fragments have been ligated to two. Acetobutylicum shuttle vectors under control of the ptb (phosphotransbutyrylase) or the thl (thiolase) promoters. Initial efforts to identify.
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They bind proteins in non-native states and assist in their proper folding. The role and regulation of the Dnakj and Groesl protein families. Acetobutylicum is not paper well understood. Orf, a is a putative repressor of the Groesl and Dnakj operons, as identified by homology to hrc, a in Bacillus subtilis. Hrc, a has been shown to operate as a negative repressor through interaction with a circe (Controlling Inverted Repeat of Chaperone Expression) element located upstream of both the Dnakj and Groesl operons. The orf, a gene was cloned from the Dnakj operon using pcr primers that amplified the structural sequence, assignment but not the regulatory sequences. The orf, a gene was ligated to an Escherichia coli/C.
Acetobutylicum shuttle vector such that orf, a is under the control of the clostridial thiolase (thl) strong promoter. The resulting plasmid (p orf, a1) was transformed into. Northern Blot analysis of the orf a overexpression strain 824 (p orf A1) was compared to that of the wild type 824 strain to verify increased transcription of orf A from p orf. A probe designed to detect the orf A gene resulted in a strong.1kb band present in the 824 (p orf A1) samples, but not in the wild type 824 samples. The same orf A probe also resulted in.8kb band present in the wild type samples, that was not present in the 824 (p orf A1) samples. The.8kb band corresponds to the expected.8kb transcript from the Dnakj operon. The fact that this transcript is absent in the orf a overexpressing strain is expected if the orf A protein is in fact acting as a repressor of the Dnakj operon.
Also, it is very important basic knowledge. Past efforts to produce tolerant strains have relied on selection under applied pressure and chemical mutagenesis, with some good results, but not consistently. We will examine metabolic Engineering (ME) and genomic approaches to determine if they can be used to construct more tolerant strains for bioprocessing. The accepted dogma is that toxicity is due to the chaotropic effects of solvents on the cell membrane. Impaired membrane fluidity and function inhibit cell metabolism, and result in cell death. We have found that in Clostridium acetobutylicum, several well-defined genetic modifications not related to membrane function impart solvent tolerance (by 40-70 percent) without strain selection.
This suggests that we need to reexamine the accepted dogma. The objective of this research is to identify genes that contribute to solvent tolerance and to use genetic modifications (involving these genes) to generate solvent tolerant strains. In view of the large number of possible genes that may be involved in determining solvent tolerance, we will use dna microarrays for transcriptional analysis. Progress Summary: Our progress is described below. Overexpression of orf a, a putative repressor of the Groesl and Dnakj operons. The major heat shock proteins Dnakj and Groesl are essential to normal cellular function.
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Department : Rohini zone 13 mpr under mplad scheme upto.05.16 Department : Engineering hq 14 mpr under mplad scheme upto.04.16 Department : Engineering hq 15 mpr under mplad scheme upto.03.16 Department : Engineering hq 16 mpr under mplad scheme upto.02.16 Department. Department : Others 18 mpr under mplad shceme upto.10.15 Department : Others 19 mpr under mplad scheme upto.09.15 Department : Engineering hq 20 mpr under mplad scheme upto.08.15 Department : Engineering hq 21 mpr under mplad scheme upto.05.15 Department : Engineering. Aggarwal, hon'ble mp (LS) upto 2013-14, edmc department : Engineering hq 48 Monthly Progress Reports under mp fund Scheme upto.07.13 in North Delhi municipal Corporation best Department : Engineering hq 49 Status Report of mla fund for the year of 2009-10 upto of North dmc. Epa grant Number: R828562, title: Metabolic Engineering of Solvent Tolerance in Anaerobic Bacteria. Institution: Northwestern University, epa project Officer: Richards, April, project Period: June 1, 2000 through may 31, 2003. Project Period covered by this Report: June 1, 2000 through may 31, 2001. Project Amount: 180,000, rFA: Technology for a sustainable Environment (1999). Rfa text, recipients Lists, research Category: Sustainability, pollution Prevention/Sustainable development, objective: Understanding solvent (and other toxic chemical) tolerance of microorganisms is crucial for the production of chemicals, bioremediation, and whole-cell biocatalysis.
Department : Engineering hq 6, mpr under mplad scheme upto.06.17. Department : Engineering hq 7, mpr under mplad scheme upto.05.17. Department : Engineering hq 8, mpr under mplad scheme upto.04.17. Department : Engineering hq 9, mpr under mplad scheme upto.03.17. Department : Engineering hq 10, mpr under mplad scheme upto.02.17. Department : Engineering hq 11, mpr under mplad scheme upto.07.16. Department : Engineering hq 12, help mpr under mplad scheme upto.06.16.
residence, and the reexamination, if required, must be scheduled no later than June 15th. Exceptions to the rule may be granted by the faculty in unusual circumstances such as extended illness or a change of research advisors, but only upon written petition by the student to the graduate studies committee prior to the end of the first term. Progress Report of mplad scheme, sNo. Document Name, date Uploaded. Report of mpr under mplads fund upto.05.18. Department : Engineering hq 2, report of mpr under mplads fund upto.01.18. Department : Engineering hq 3, mpr under mplads fund upto.12.17. Department : Engineering hq 4, mpr under mplad scheme upto.07.17. Department : Engineering hq 5, mpr under mplad scheme upto.08.17.
The written research progress report required for admission to candidacy must be submitted to the faculty before the end of the second term of the second year in residence. The report is to be based upon the PhD research project and is expected to exhibit a professional quality of exposition and should outline the research problem, the progress of the student at the time of writing, and the areas proposed for future study. It is also important that the report provide clear evidence of the student's understanding of the motivation for the research problem and its relevance to the general research area, as well as an appreciation of relevant techniques and methodology. The student should strive for a precise and concise format. Normally, the text itself should not exceed 20 double-spaced, typewritten pages. The research progress report needs to be approved by an oral examination committee consisting of no less than three faculty members, including the research advisor, and at least one additional member of the Chemical Engineering faculty. The choice of the oral exam committee will be made by the student and his/her research advisor, and must be approved by the Option is the responsibility of the student to obtain approval from each proposed committee member for the date and time of the. The list of the recommended committee members must be submitted at the same time as the research progress report, and the exam is normally scheduled thesis within two weeks of submitting the report. However, reports submitted within the last week prior to final course exams will normally be examined within the first two weeks of the subsequent quarter.
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Adam Colgate, how to read an Earnings Report. Knowing how to read an earnings report summary gives an analyst or investor a bottom line perspective into the finances and profitability of a company. Publicly traded companies must file a quarterly report to the securities and Exchange commission. Graduate Program in Chemical Engineering 1st year 2nd year 3rd year 4th year 5th year, seminars, prior to admission to candidacy for the PhD degree, a student is required to pass the candidacy examination. This exam consists of two parts: a research progress report and an oral examination based primarily upon the content of the report. A student who fails to pass the candidacy exam according to the timetable and procedures described below will not be permitted to register in any subsequent terms, except for the purpose of completing work toward the ms degree, with the permission of the graduate studies. Graduate Office forms, option Committee and Date Approval Form.