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Molecular characterization of Bacillus thuringiensis and contruction of a recombinat bacteria

Molecular characterization of Bacillus thuringiensis and contruction of a recombinat bacteria. Frank Jeyson Hernández. Introduction:. Bacillus thuringiensis (Bt). Bt is an insecticidal bacterium, marketed worldwide for control of many important plant pests but also mosquito larvae.

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Molecular characterization of Bacillus thuringiensis and contruction of a recombinat bacteria

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  1. Molecular characterization of Bacillus thuringiensis and contruction of a recombinat bacteria Frank Jeyson Hernández

  2. Introduction: Bacillus thuringiensis (Bt) Bt is an insecticidal bacterium, marketed worldwide for control of many important plant pests but also mosquito larvae. Bt products represent about 1% of the total ‘agrochemical’ market (fungicides, herbicides and insecticides) across the world.

  3. Genes and Toxicity The extraordinary diversity of B. thuringiensis strains and toxins is in part through a high degree of genetic plasticity. Most of the toxin genes appear to reside on plasmids, which are conjugative in nature. The generation of recombinant bacteria simply by electroporation, or by homologous recombination using an exogenous cry gene and a bacterium which has a better culturing properties of recombinant bacteria, e.g. E. coli, will also be investigated.

  4. Genes and PCR B. thuringiensis strains will be characterized by the help of genotype for PCR technique. The polymerase chain reaction (PCR) is a rapid way of amplifying (duplicating) specific DNA sequences. Method was devised by Kary Mullis of Cetus Corporation, Emeryville

  5. Construction of the Plasmid • Selected the plasmid having more interesting genes, to incorporate few new genes. • The ideal plasmid is the following combination:cry 4Aa1, cry4Ba4, cry 11Aa2, cyt 2Ba and selective marker gene(e.g. Antibiotic resistence). • Found one native plasmid of Bacillus thuringiensis (pTX14-3) that contain 3 interesting genes and was modified using restriction enzymes to add two new genes (kanamicina resistence and cry 11Aa2).

  6. FOREING DNA EcoRI Region of Interest EcoRI EcoRI Sticky ends Hibridization + DNA Ligase RECOMBINANT DNA Restriction enzyme The foreign fragment of DNA is added to the plasmid or viral DNA as shown to make a recombinant DNA molecule. This technique of DNA cloning is the basis for the entire field of recombinant DNA technology.

  7. cry 4Aa1 cry 11Aa2 cry 4Ba4 Kamres cyt 2Ba Ori native native native Recombinant Plasmid This plasmid is incorporated in a strain of E. coli.

  8. DNA Insertion RECOMBINANT DNA E. coli Bacterial Chromosome Bacterial platted on medium + antibiotic (Kanamicine) CLONING Only Bacteria containing Reconbinant DNA grow DNA Purification CULTURE CLONE

  9. References http://helios.bto.ed.ac.uk/bto/microbes/bt.htm http://www.bio.ic.ac.uk/research/djwright/ http://likya.iyte.edu.tr/ar_fon_proje/digerduyurular/YenidunyaGunesING.htm

  10. Thanks !!

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