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

2. 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. Introduction: Bacillus thuringiensis (Bt)

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. 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. EcoRI FOREING DNA Region of Interest Sticky ends Hibridization + DNA Ligase RECOMBINANT DNA

7. Ori cry 4Aa1cry 11Aa2cry 4Ba4cyt 2BaKam res native This plasmid is incorporated in a strain of E. coli. Recombinant Plasmid

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

9. 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/Yenidunya GunesING.htm References