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Preparation of a genomic DNA library

   

Added on  2020-04-15

19 Pages3826 Words84 Views
Running head: GENETIC ENGINEERINGCourse Code:GENETIC ENGINEERINGName of the Student:Name of the Partner:Name of TA:Lab Section:Report Submission Date:

1GENETIC ENGINEERINGAbstractThepurpose of the study was to prepare a genomic DNA library. A genomic DNA library is created inorder to identify the DNA fragments or genes that are of interest to a researcher. The subsequent genecloned in the vector can be expressed to obtain the desired protein product. The genomic DNA librarywas created by using the genomic DNA of Bacillus subtilis. The genomic DNA was isolated from theorganism and its concentration and purity were determined. Similarly, the plasmid DNA pUC18 wasused as the vector, which is required during cloning of the genomic DNA fragments into the vector.The resulting genomic DNA and plasmid DNA were digested with two different restriction enzymes,subsequently ligated and transformed into the Escherichia coli DH5α cells on X-gal/IPTG mediumcontaining plates. The ratio of blue to white colonies obtained were 3:8. The blue colonies did notcontain the inserts obtained from genomic DNA, while the white colonies carried the recombinantDNA having desired inserts obtained from genomic DNA ligated to the vector pUC18. Therecombinant plasmids were isolated from the desired white colored clones and subjected to doubledigestion with the same restriction enzymes, which were used to digest the plasmid and the genomicDNA. Double digestion yielded the desired insert and the vector, which in this case is pUC18. Singledigestion of recombinant plasmid and vector showed the desired shift due to the differences in sizeresulting from the presence of the insert in the plasmid DNA. Thus, cloning was successful as thedesired recombinant clones carrying the genomic DNA insert was obtained.

2GENETIC ENGINEERINGTable of ContentsIntroduction......................................................................................................................................3Results..............................................................................................................................................4Lab 3 results.................................................................................................................................4Lab 4 results.................................................................................................................................5Lab 5 results.................................................................................................................................5Discussion........................................................................................................................................7Reference List................................................................................................................................10Appendix........................................................................................................................................12

3GENETIC ENGINEERINGIntroductionBacillus subtilis is a Gram positive and rod shaped microorganism. It produces dormant andheat resistant spores and is non-pathogenic (Leggett et al., 2012). The genomic DNA of B. subtilis iscircular and is approximately 42,14,630 base pairs, with a GC content of 43.5% and encodes 4100proteins (van Dijl & Hecker, 2013). It is a soil bacterium and is used for the production of manyindustrial products like commercial enzymes like proteases and amylases, vitamins like riboflavin,supplements like poly gamma glutamic acid, flavoring agent ribose, industrial nucleotides, amongothers (Singh et al., 2016). It’s genome can be easily manipulated and as a result can be used in geneticengineering. It is the best characterized of among all the Gram-positive bacteria species having low GCcontent. Genomic DNA libraries are collections of genomic DNA sequences from an organism. Thistype of library consists of all the gene sequences present in the genome of the organism. Each cloneconsists of at least on one copy of the DNA sequences or genes present in the genome. The wholegenome of the organism is represented by a set of genes or DNA segments inserted into a vector DNAmolecule. Genomic library serves many purposes. It helps to determine the whole genome sequence ofan organism, helps in the study of the functions of the genes or regulatory sequences; it helps todetermine the presence of any genetic alterations or mutations, particularly in cancer tissues, helps inexpression of genes that encodes proteins of industrial or commercial importance. It can also help inthe production of novel pharmaceutical products (Rohland & Reich, 2012). Genetic engineering is the direct manipulation of the genetic organization of an organism usingthe technique of recombinant DNA technology. It helps in the transfer of genes from one organism andits subsequent expression in another organism. Apart from inserting new genes, genetic engineering

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