Progress 09/01/12 to 08/31/13
Outputs
Target Audience: Scientists and faculties, graduate and undergraduate students, adminastrators, and stakeholders. These people were reached by providing training to graduate and undergraduate students; making presentations at regional and national conferences; presenting the research work to adminastrators at various levels and stakeholders to emphasize the importance of renewable bioenergy. Changes/Problems: The following technical procedures have been changed. In the original proposal, we propose to use metagenome library functional screening approach to isolate cellulosic genes. After various attempts, we realized that it is very difficult to find useful genes by taking this approach which could be due to the complex enzyme system required to degrade cellulose. Then the next generation sequencing approach was used, and about 200 cellulosic genes were found in the annotated genemoes. We are cloning those genes from the geneome DNA. This trial procedure has delayed the progress of the project. While conducting this project, we have isolated an Bacillus bacteria showing cellulosic activity on plate assay. We have confirmed the cellulosic activty, and identifed genome sequences and structured of this isolate. In addition, this bacterial strain seems to produce a useful compounds. We are in the process of confirming this compounds which may not be related to the production of bioethanol but it has very important industrial use. With the discovery of this bacterial strain, the project has expanded into a new direction. What opportunities for training and professional development has the project provided? One master student has graduated from this project in Summer, 2013; In Fall, 2013, one Ph.D. and one master student were recruited to continue the research project; PI has attended a DOE workshop on using the system for metagenome analysis in 2013; Graduate students attended 2013 summer proteomics internship at the USDA/ARS laboratory on Cornell campus and the proteomics workshop and confocal microscopy training at Cornell University; Undergraduate students have been trained on bioinformatics, PCR and basic laboratory analysis. Students have received the following awards: Hui Li. 2013. The first award for oral presentation in the Category of Sciences at TSU 2013 Research Symposium; Hui Li. 2013. The third award for oral presentation at the 17th Biennial Research Symposium (ARD) Conference. March 9-15,Jacksonville, FL. How have the results been disseminated to communities of interest? Desemination of the research findings is achieved by presentations by scientists and students at regional and national and international conferences. The annotated metagenome sequences are up-loaded into DOE database and will release to the public pending on publication and completion of the patent application process. The folloiwing are examples of presentations made by the students: Li, H, Zhou, S., Johnson, T. , Vercruysse K.P. 2013. Genome structure and protein expression for cellulosic activities of a new bacterial strain. 17th Biennial Research Symposium (ARD) Conference. March 9-15, Jacksonville, FL. Li Hui, Johnson Terrace, Koen Vercruysse, Suping Zhou. 2013. Characterization of a bacterial strain for its activity in degrading cellulose and producing biopolymer material. 33rd Annual University-Wide Research Symposium. TSU, March 2-7, Nashville, TN. Peter Nveawiah-Yoho, Jing Zhou, Marsha Palmer, Roger Sauve, and Suping Zhou1, Kevin J. Howe, Tara Fish, and Theodore W. Thannhauser. 2013. Identification of proteins for salt tolerance using a comparative proteomics analysis of tomato accessions with contrasting salt tolerance. J. Amer. Soc. Hort. Sci.138:1–13. In print. What do you plan to do during the next reporting period to accomplish the goals? 1. Cloning and selection of cellulollytic genes based on the enzymatic activity of recombinant proteins; 2. Making multiple-gene vectors; 3. Publish the resutls; 4. Transfer goat's rumen cellulolytic genes into the Bacillus isolate to enahnce its activity on natural biomass products; 5. To test various renewable products in attempt to develop a system to grow the bacteria without using commercial media such as LB.
Impacts
What was accomplished under these goals?
1). Functional screening of metagenomic libraries could not find clones showing cellulase activtiy. We then used the next generation sequencing approach to identify those genes. The metagenome sequences were assembled and annotation was done using the pipeline developed by DOE Join Genome Institute. 2). We have isolated a Bacillus cereus strain which was isolated while conducting this project. This bacterium also has activity in degrading cellulosic substrates. The secretive proteome of the bacteria was identified using nano-LC-MS proteomics analysis. Gel permeation chromatography was used to validate cellulosic activity on soluble and insoluble substrates; Interesting gene sequences with putative functions in the pathway of converting cellulose to glucose were identified from the annotated genome sequences. 3). PCR cloning of interesting genes from metagenome DNA and Bacillus genome DNA are undergoing together with preparation of expression constructs to select genes with higher cellulolytic activities and developing a system to convert biomass into glucose. 4). The products were submitted for patent application. However, it was returned due to the new patent law on natural products. We are working on developing a patentable products.
Publications
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2013
Citation:
Hui Li. 2013. Characterization of A Bacterial Strain With Putative Cellulosic Activity. Master's thesis, Tennessee State University.
Li, H, Zhou, S., Johnson, T. , Vercruysse K.P. 2013. Genome structure and protein expression for cellulosic activities of a new bacterial strain. 17th Biennial Research Symposium (ARD) Conference. March 9-15, Jacksonville, FL.
Li Hui, Johnson Terrace, Koen Vercruysse, Suping Zhou. 2013. Characterization of a bacterial strain for its activity in degrading cellulose and producing biopolymer material. 33rd Annual University-Wide Research Symposium. TSU, March 2-7, Nashville, TN.
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Progress 09/01/11 to 08/31/12
Outputs
OUTPUTS: During this period, we have incorporated the next generation sequencing and proteomics technology into the project. A new Bacillus bacterial strain with putative cellulosic activities was isolated, and the whole genome sequence was analyzed in collaboration with Cornell University, and then the sequence was assembled (so far only partially) in collaboration with the Pittsburg Supercomputer Center. Then proteins excreted by the bacteria into the growth media was characterized in collaboration with Dr. Thannhauser's lab in USDA-ARS/Cornell. These results will guide the next step for cloning interesting genes and characterization of potential use of the bacterial strain and the genome sequences in biomass conversion and production of bio-materials. For project disseminations, DNA sequences cloned from goat's rumen bacteria have been deposited in the NCBI GenBank database to be accessible to interested parties worldwide. To share the research results with scientific communities, the PI and graduate students have presented the research results on international conferences. On TSU campus, the disseminations activities include presentation by the PD to graduate classes, and student presentations during the university- wide research symposium. The project was included in a presentation given to a Research Seminary Graduate class of Fall 2011in the Department of Biology of TSU. PARTICIPANTS: Puchana Rysard, Langston University, Charles Lee, Bioproduct Chemistry and Engineering Research USDA,ARS, Theodore Thannhauser, Plant, Soil and Nutrition Research Unit, USDA,ARS, Alex Ropelewski,Pittsburg Supercomputer Center TARGET AUDIENCES: Students, scientific community and general public PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We have isolated a Bacillus bacterial clone with putative cellulosic activities on cellulose medium plate. Using next generation sequencing technique, the whole genome was sequenced and assembled. Then the assembled contigs were used to develop a protein database. Analysis of the secretome proteins of the bacteria have identified several proteins relevant to degradation of cellulosic materials such as glucanase A (Cellulase A), endoxylanase, endo-beta-1,4-xylanase precursor , endo-beta-1,4-xylanase, pectate lyase, cellulase, glycoside hydrolase, xylan 1,4-beta-xylosidase. These DNA and protein sequences will be used to prepare recombinant gene construct to produce those enzymes. The same technical procedure are used in identification of cellulolytic genes from goat's rumen microbial metagenomes. During this one year period, several graduate students and undergraduates have received training in different phases of the project. This project has provided the venue for establishing collaboration with Pittsburg Supercomputer Center and acquiring access to the facilities and technical support from the expertise in that center, and collaboration with USDA-ARS scientists to receive their support and have access to the on-site facilities. These partnerships are very important for training the current students and carrying out this project, additionally these resources are very valuable for continuation and expansion of the research and student training at TSU.
Publications
- Zhou, J., Copeland, B., Zhang, C., Liu, Z., Bhatti, S., Sauve, R. and Zhou, S. 2011. Identification of prokaryotic organisms in goat rumen based on metagenomic DNA sequences. Journal of research in Biology 6: 451-455.
- Li, H., Zhou, S., Copeland, B., Johnson, T. and Sauve, R. 2012. Characterization of a bacterial strain with putative cellulosic activity. Plant and Animal Genome XX Conference. San Diego, CA, Jan.14-18.
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: 1. Metagenomic DNA and RNA libraries are being prepared to identify genes that are associated with cellulosic activities. However, no DNA sequences for cellulases were found in the metagenomic DNA library. New library will be constructed and screened using strategies other than Congo Red staining method. 2. Recruited graduate students to be trained in biotechnology PARTICIPANTS: Brian Copeland, graduate student TARGET AUDIENCES: A manuscript has been submitted for journal publication, and gene clone sequences deposited in Genbank database. Those information will be shared by the scientifc community as well as the public. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
One bacterial clone has been isolated, which shows hydrolytic activity on various cellulosic substrates. We are confirming the enzymatic activity of the secreted proteins from the bacterial culture using glucose assay. Total RNA was isolated from goat's rumen fluid. After removal of rRNA, the RNA extract will be prepared for a whole genome sequence ananlysis to identify useful genes from this resource. A metageomic library was made from goat's rumen bacteria and sequenced. A manuscript has been sumitted for journal publication. Twenty gene clones have been deposited in the Genbank database. One graduate student is receiving training on this project.
Publications
- No publications reported this period
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