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Volume: 2, Issue: 6, Nov-Dec, 2014
DOI: 10.7324/JABB.2014.2602

Research Article

Amplification and cloning of mcrAgene from enriched consortia of Methanogens

Rachana Chandragupta Acharya, Usha Mukundan

Abstract

Methanogens were enriched using anaerobic culture conditions. The gDNA extracted from the enriched consortia was used for amplification of mcrA (methyl coenzyme reductase A) gene. The temperature of annealing was standardized for amplification. The amplified gene was cloned into cloning vector and transformed into E.coli DH5α cells. Screening of recombinant transformants was done using Blue-White selection. The vector containing amplified gene was sequenced. The sequencing results showed that the gene had 97% homology with mcrA gene from Methanoculleus bourgensis. Thus procedure for mcrA gene amplification from enriched Methanogen consortia was standardized and its marker assisted identification using bioinformatics tools was carried out.

Keywords: Anaerobic culture, temperature of annealing, PCR amplification, marker assisted identification.

References

1. Gracia J.L., Patel B.K.C., and Ollivier B. Taxonomic, Phylogenetic and Ecological Diversity of Methanogenic Archaea. Anaerobe. 2000; 6, 205-226.

2. Horwitz J.P., Chua J., Curby R. Tomson A,J., DaRooge M.A., Fisher B.E., Mauricio J., and Klundt. Substrates for cytochemical demonstration of enzyme activity.1. Some substituted 3-indolyl-D-glycopyranosides. Journal of Medical Chemistry. 1964; 7 574-575.

3. Luton, P.E., Wayne, J.M., Sharp, R.J., Riley, P.W. The mcrA gene as an alternative to 16S rRNA in the phylogenetic analysis of methanogen population in landfill. Microbiology. 2002; 148, 3521\—3530.

4. Ferry J. G. Methanogenesis: Ecology, physiology, biochemistry & genetics. Chapman & Hall microbiology series. New York: Chapman & Hall. 1993.

5. Collins G, Woods A, McHugh S, Carton M W, O'Flaherty V. Microbial community structure and methanogenic activity during start-up of psychrophylic anaerobic digesters treating synthetic industrial waste water. FEMS Microbioly E.coli. 2003;46:159\—70.

6. Bauer C, Korthals M, Gronauer A, Lebuhn M.. Methanogens in biogas production from renewable resources \—a novel molecular population analysis approach. Water Science and Technology. 2008; 58: 1433 \—9.

7. Rastogi G., D.R. Ranade, T.Y. Yeole, A.K. Gupta, M.S. Patole and Y.S. Shouche. Molecular analyses of methanogen diversity associated with cattle dung. World journal of microbiology and Biotechnology. 2008; 24: 2973-2979.

8. Ni SS., Boone D. R. Isolation and characterization of a dimethyl sulfide-degrading methanogen, Methanolobus siciliae HI350, from an oil well, characterization of M. siciliae T4/MT, and emendation of M. siciliae. International Journal of Systematic Bacteriology. 1991; 41, 410\—416.

9. BLAST. Available from - http://www.ncbi.nih.gov/

10. CLUSTAL W. Available from- http://www.ebi.ac.uk/Tools/clustalw2/index.html.

11. Vogels G.D., Keltjens J.T., Van der Drift, C. Biology of Anaerobic Microorganisms, Alexander J.B. Zehnder (Ed.) John Wiley & Sons, New York, 1988; 707-770.

12. Singh K. M., Paresh R. P., Subhash Parnekar, Ajai K. T, Umed R. Prakash G. K. Dharmshi N. R. Chaitanya G. J. and Ramesh K. K. Methanogenic Diversity Studies within the Rumen of Surti Buffaloes Based on Methyl Coenzyme M Reductase A (mcrA) Genes Point to Methanobacteriales‖. Polish Journal of Microbiology. 2010, 59(3), 175-178.

How to cite this article:
Rachana Acharyaa and Usha Mukundan. Amplification and cloning of mcrAgene from enriched consortia of Methanogens. J App Biol Biotech. 2014; 2 (06): 007-009. DOI: 10.7324/JABB.2014.2602

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