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Santosh Thapa

Phd in Biological Science

Tennessee State University, Nashville TN, 37209

Thesis : Functional characterization of cellulolytic enzymes from metagenome in goat’s rumen

Anticipated date for completion : Fall 2018.

(Advisor: Suping Zhou; Committee Members: Roger J. Sauve, Fur-Chi Chen, Samuel Nahashon, and Terrance L. Johnson)

Project Summary

The paucity of enzymes that efficiently deconstruct plant polysaccharides represents a major bottleneck for industrial-scale conversion of cellulosic biomass into biofuels. The cellulosic bioconversion into fermentable sugar is a complex process and requires the synergistic action of endoglucanases, exoglucanases and β-glucosidases. These enzymes are secreted by the ruminal microbes in ruminant animals such as goats. To be able to tap into those resources is expected to have a significant impact on cellulosic biofuel production, but a majority of those microbial species are not culturable under laboratory conditions (using laboratory culture techniques). This study was performed to develop molecular approaches for using these genome resources. Metagenome DNA was extracted and sequenced using next-generation sequencing.  Following assembly and annotation, 14 gene clones encoding for cellulases, hemicellulases and xylanases have been cloned from the metagenome.  PCR primers were designed against the assembled gene sequences, and PCR reactions were performed using metagenome DNA as the template.  The amplified PCR products were cloned using TA cloning strategy.  Gene sequences were confirmed using Sanger sequencing and submitted to NCBI database. Recombinant proteins of these cellulase genes will be produced to select for highly effective enzymes for biomass degradation. Outcomes from this project will increase the enzyme species for producing value-added products from cellulosic biomass materials.

Current Responsibilities

• Develop skills in identification and cloning of cellulolytic enzymes from metagenome in goat’s rumen.
• Design PCR primers to amplify the gene of interests using metagenome DNA as the template.
• Apply molecular genomic approach, fundamentally the use of PCR coupled with gel-electrophoresis, the different gene cloning strategies and bio-informatics tools for the production of hydrolytic novel enzymes.
• Develop tools and protocols (e.g. run PCR, gel electrophoresis, ligation and transformation, protocol for plasmid DNA extraction) and dissemination of findings through publications, collaboration reports and conference presentations.
• Prepare scientific reports, present results and progress reports in international/national collaboration meetings and analysis workshop.

Master in Agriculture Science                                                                                                                2013 - 2015

Tennessee State University, Nashville TN, 37209

Thesis : Identification and Cloning of genes encoding for cellulolytic enzymes from metagenome in goat’s rumen

Summer Activities 2015:

Internship                                                                                                                                Aug 04 – Aug 14 (2015)

Plant, Soil and Nutrition Research, USDA, Cornell University, Ithaca, NY.

• Worked on Itraq (isoberic tags for relative and absolute quantitation) labelling.
• Trained on Two dimensional gel electrophoresis.

Scientific Research Sample Collection                                                                         Aug 29, 2015-Sep.07, 2015

• Study Title: Lignocellulolytic Bacteria and Fungi isolated from hydrothermal vegetative environments.
• Study#: YELL-06074
• Permit#: YELL-2015-SCI-6074

Presentations

• Cloning and Characterization of Genes Encoding Cellulolytic Enzymes Screened From Goat Rumen Metagenome (Poster Presentation).  Santosh Thapa, Hui Li, Sarabjit Bhatti, Suping Zhou. The Seventh International Conference on Environmental Science and Technology. Houston, Texas, USA. June 07-10, 2016(Merit Award).

• Cloning Of Genes Encoding For Cellulolytic Enzymes From Metagenome In Goat’s Rumen (Poster Presentation). Santosh Thapa, Hui Li, Sarabjit Bhatti, Suping Zhou. The Seventh International Conference on Environmental Science and Technology. Houston, Texas, USA. June 9-13, 2014 (Merit Award).

• Goat Rumen Metagenome Extraction, Annotation and Screening for Cellulases and Hemi-cellulases Genes (Poster Presentation).  Santosh Thapa, Hui Li, Sarabjit Bhatti, Suping Zhou. Thirty-Sixth Annual University Wide Research Symposium. Tennessee State University, Nashville, TN. March 31-April 04, 2014.

• Identification and Cloning of Cellulolytic Genes From Metagenome in Goat’s Rumen (Oral Presentation) at Dean Seminar Series, College of Agricultural, Human and Natural Sciences. Tennessee State University. November 21, 2013 at Agriculture Information and Technology Centre, Tennessee State University, Nashville, TN.

• Cellulolytic Genes Cloning from Goat’s Rumen through Metagenomic Approach (Poster Presentation). ).  Santosh Thapa, Hui Li, Sarabjit Bhatti, Suping Zhou. Thirty-Seventh Annual University Wide Research Symposium. Tennessee State University, Nashville, TN. March 31-April 04, 2015.

 Achievements:

• Fouteen bacterial novel gene sequences have been cloned and so far five novel bacterial gene sequences have been  submitted to the NCBI database under the following accession number:

Ongoing Research:

• Cloning of remaining genes.
• Expression and purification of recombinant proteins.
• Enzyme activity assay on  various  substrates for cellulase, and xylanase activities;

Honors and Awards:

• Third Place Award in Poster Presentation in The Seventh International Conference on       Environmental Science and Technology. Houston, Texas, USA. June 07-10, 2016.
• Second place for his poster entitled “Cloning of genes Encoding for Cellulolytic Enzymes from Metagenome in Goat’s Rumen”, presented during the 7th International Conference on Environmental Science and Technology 2014 in Houston, Texas.