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Bioinformatics programs in the teaching lab

Bioinformatics programs in the teaching lab. BIOL 5238 Molecular Biology laboratory (one 4-hr lab period/wk). Current syllabus :. Week topic Prepare media and plate out bacteria V. fisheri gDNA preparation pt 1 V. fisheri gDNA preparation pt 2 pGEM midiprep preparation

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Bioinformatics programs in the teaching lab

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  1. Bioinformatics programs in the teaching lab BIOL 5238 Molecular Biology laboratory (one 4-hr lab period/wk) Current syllabus: • Week topic • Prepare media and plate out bacteria • V. fisherigDNA preparation pt 1 • V. fisherigDNA preparation pt 2 • pGEMmidiprep preparation • quantitate/qualitate DNA and begin digestion • Set up ligations • Prepare electrocompetent cells and electroporate • MinipreppGEM::lux candidates and analytical digestions • PCR amplificaiton ofluxA • and clean-up DNA for sequencing • Subcloning of luxA into pTOPO and tranformation • Southern blot analysis • qRT-PCR analysis • Bioinformatics • SDS-protein analysis

  2. Bioinformatics programs in the teaching lab BIOL 5238 Molecular Biology laboratory (one 4-hr lab period/wk) NEW Current syllabus: • Week topic • Prepare media and plate out bacteria(begin isolation of environmental bioluminescent bacteria) • V. fisherigDNA preparation pt 1 • V. fisherigDNA preparation pt 2 • pGEMmidiprep preparation • quantitate/qualitate DNA and begin digestion • Set up ligations (introduction to BLAST searches – HW: design universal luxA PCR primers) • Prepare electrocompetent cells and electroporate • MinipreppGEM::lux candidates and analytical digestions • PCR amplificaiton ofluxA(from transformantand environmental microbe [16S rRNA]) • and clean-up DNA for sequencing • Subcloning of luxA into pTOPO and tranformation(identification of unknown, luxA alignment) • Southern blot analysis (PFGE with V. fisheriand unknown pt 1), Expasy analysis of Lux proteins • qRT-PCR analysis (PFGE with V. fisheriand unknown pt 2), Lux A alignment, phylogenetic tree • Bioinformatics (PFGE with V. fisheriand unknown pt 3), Lux A Consurf • SDS-protein analysis (student presentations – Expasy tools) V. harveyiLuxA

  3. Bioinformatics programs in the research lab M. abscessus M. aichiense M. alvei M. arupense M. aubagnense M. asiaticum M. aurum M. avium subsp. avium M. barassiae M. bolletii M. cookii M. chelonae M. celatum M. chitae M. chubuense M. conceptionense M. chlorophenolicum M. chimaera M. crocinum M. diernhoferi M. fortuitum M. farcinogenes M. flavescens M. fortuitumacetamidolyticum M. gastri M. gilvum M. gordonae M. genavense M. goodii M. hiberniae M. houstonense M. intracellulare M. intermedium M. isibruicum M. kuopiense M. kumamotonense M. lentiflavum M. madagascariense M. marinum M. malmoense M. mageritense M. montefigrense M. moriokaense M. mucogenicum M. neoaurum M. neworleansense M. neglecticum M. nonchromogenicum M. paraffinicum M. parafortuitum M. parascrofulaceum M. peregrinum M. porcinum M. rhodesiae M. salvoniae M. scrofulaceum M. senegalense M. septicum M. setense M. simiae M. sphagni M. smegmatis M. solinskyi M. szulgai M. terrae M. triplex M. tusciae M. vaccae M. xenopi

  4. Bioinformatics programs in the research lab Project 1: What mycobacteria are in the bog? Mean sequence similarity* DNA sequence 16S rRNA 96.6% rpoB 91.3% Development of Lsr2 as a molecular chronometer hsp65 91.1% dnaJ 80.4% M. gastri M. kansasii M. mucogenicum M. phocaicum * 56 Mycobacterial species sequenced 100% identity for 16S rRNA Yamada-Noda et al. 2007 Lsr2 (Conserf) http://consurf.tau.ac.il/results/1299874910/output.html

  5. Bioinformatics programs in the research lab Project 2: Do any bog mycobacteriasporulate? Sporulation in mycobacteria. Ghosh J, Larsson P, Singh B, Pettersson BM, Islam NM, Sarkar SN, Dasgupta S, Kirsebom LA. Proc Natl Acad Sci U S A. 2009 Jun 30;106(26):10781-6. Do mycobacteria produce endospores? Traag BA, Driks A, Stragier P, Bitter W, Broussard G, Hatfull G, Chu F, Adams KN, Ramakrishnan L, Losick R. Proc Natl Acad Sci U S A. 2010 Jan 12;107(2):878-81. Growth, cell division and sporulation in mycobacteria. (2010) Singh B, Ghosh J, Islam NM, Dasgupta S, Kirsebom LA. Antonie Van Leeuwenhoek. 2010 Aug;98(2):165-77 65oC, 15 min wet • Growth on solid media • PCR (dnaJ, lsr2) • Metagenomic sequencing • Comparison of sequences • Identify acid-soluble proteins • BLAST search for SASPs dry wet dry

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