Molecular Identification of DMS-Producing Bacteria Isolated from Dissertation

Molecular Identification of DMS-Producing Bacteria Isolated from Marine Algae - Dissertation Example Michael Steinke, for his unfailing and relentless support, and insightful remarks. His guidance in the conception and interpretation of this study granted me the confidence and multiplied my abilities for the successful execution of this project. I am grateful to the University, especially to the Department of Biosciences for enabling me in bringing this study to realization by providing me with all the necessary resources and support. I am greatly indebted to the University, my supervisor and colleagues, without whom the completion of this dissertation would have been a highly daunting task if not totally impossible. Contents Abbreviations 8 Abstract 10 1. Introduction 11 1.1 Distribution of DMSP in upper and lower photic zones 12 1.2 Microbial breakdown of DMSP and production of DMS 12 1.2.1 Microbes involved in DMSP breakdown 12 1.2.2 Mechanism of conversion of DMSP to DMS 16 1.2.3 Genes, proteins, promoters and gene regulation 18 1.3 Environmental significance of DMS and its role in global climate 25 1.4 Importance of identification, characterisation and phylogenetic analysis of DMS producing bacteria 26 2. Materials and methods 28 2.1 Isolation and growth of DMS producing bacteria 28 2.2.1 Preparation of growth media 28 2.2.2.1 Chemicals and reagents required 29 2.2.2.2 Preparation of M9 media of normal salinity with glucose as a carbon source 29 Rami Abdullah Aldagrer September 9th, 2011 Contents UNIVERSITY OF ESSEX 1 DEPARTMENT OF BIOLOGICAL SCIENCES 1 MSc. DEGREE IN BIOTECHNOLOGY 1 Acknowledgements 2 Contents 2 Abbreviations 8 Abstract 10 1. Introduction 11 1.1 Distribution of DMSP in upper and lower photic zones 12 1.2 Microbial breakdown of DMSP and production of DMS 13 1.2.1 Microbes involved in DMSP breakdown 13 1.2.2 Mechanism of conversion of DMSP to DMS 16 1.2.3 Genes, proteins, promoters and gene regulation 18 1.3 Environmental significance of DMS and its role in global climate 24 1.4 Importance of identification, characterisation and phylogenetic analysis of DMS producing bacteria 26 2. Materials and methods 28 2.1 Isolation and growth of DMS producing bacteria 28 2.2.1 Preparation of growth media 28 2.2.2.1 Chemicals and reagents required 29 2.2.2.2 Preparation of M9 media of normal salinity with glucose as a carbon source 29 2.2.2.3 Preparation of M9 media of normal salinity with DMSP as a carbon source 29 2.2. 2.4 Preparation of M9 media without any carbon source of normal salinity 29 2.2.2.5 Preparation of M9 media of high (32 N) salinity 30 2.2.3 Inoculation into three different media to identify DMSP utilizing bacteria 30 2.2.4 Gram staining 31 2.2.5 Spectrophotometric analysis of growth and calculation of specific growth rate and doubling time 31 2.2 Extraction of DNA 31 2.2.1 Chemicals and materials required for the CTAB method of DNA extraction: 32 2.2.2 Preparation of reagents 32 2.2.3 Methodology of DNA extraction 33 2.2.4 Verification of extracted DNA 34 2.3 Amplification of 16s rRNA using PCR 34 2.4 Purification of PCR products 35 2.5 Identification of bacteria and phylogenetic analysis 35 3. Results 37 3.1 Observation of growth in different media 37 3.1.1 Growth in M9 media with DMSP 37 3.1.2 Growth in M9 media with glucose 38 3.1.3 Growth in M9 media without any carbon source 39 3.2 Results of gram staining 40 3.3 Plotting of growth curve 41 3.4 Calculation of specific growth rate and doubling time 44 3.4.1 Specific growth rate of bacteria in M9 media with glucose 45 3.4.1.1 Specific growth rate of B3B 45 3.4.1.2 Specific growth rate of B2B 46 3.4.1.3 Specific growth rate of B2A 46 3.4.2 Calculation of doubling time of bacteria in M9 media with glucose 47 3.4.3 Specific growth rate of bacteria in M9 media with DMSP 47 3.4.3.1 Specific growth rate of B3B 47 3.4.3.2 Specific growth rate of B2B 48 3.4.3.3 Specific growth rate of B2A 48 3.4.4 Calculation of doubling time of bacteria in M9 media with DMSP 49 3.4.5 Summary of specific growth rate and generation time 50 Specific growth rate bacteria in M9 media with glucose and M9 media with DMSP 50 Doubling time of bacteria in M9 media with glucose and M9 media with DMSP 50 3.4.6 Analysis of variance between the growth rates in two different media 51 3.4.7 Analysis of variance in doubling time 52 3.2 Results of DNA extraction 53 3.3