Experiment #7.docx - Bacterial transformation and protein...

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Bacterial transformation and protein purification CHM 6610 Introduction : A. Bacterial Transformation: 1
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In this experiment, a bacterial strain was transformed with a plasmid that contains the coding sequence for the Green Fluorescent Protein (GFP) and plates the transformed cells on plates containing antibiotic and determined the transformation efficiency and GFP expression. 3 A plasmid (small extra-chromosomal DNA) contains an origin of replication and coding sequences for several proteins from E. coli. That can be used as a vector to insert the gene of interest into the plasmid vector. This vector was taken up by living cells in order to replicate and transcribe the specific gene. This can be accomplished by splicing the vector and gene then the gene is ligated into the spliced vector. Thus, this vector is referred to as recombinant DNA. If this recombinant DNA is successfully transferred into a living organism cells then it will be replicated many times and thus leads to multiple copies of recombinant DNA. Restriction endonucleases are enzymes used to splice the gene and the plasmid together. They evolved as a defense mechanism that cleaves DNA at specific sequences to stop the infection of the cell. Therefore, the cell prevents the endonucleases from cleaving its own DNA by methylating the DNA sequences that are recognized by the restriction endonucleases. So, a plasmid is cleaved with endonucleases that recognized a specific pattern, and the gene of interest is also cleaved at the ends with same endonucleases results in sticky ends that contains unpaired bases where the enzyme cut and thus the gene and the plasmid can be pared. 1 In this experiment, CaCl 2 was used to disrupt the E.coli membrane, pGlo plasmid (contains ORI, β-lactamase gene, ara C – arabinos operon genes, and the GFP gene) was used to produce an protein known as Green Fluorescent Protein (GFP). GFP is a protein that is coded by fluorescent jelly fish Aquorea Victoria. GFP has a barrel like structure composed of 238 amino acids with 11 β strands on outside of cylinders that are very stable. pGlo plasmid, GFP structure, and the mechanism of GFP prodcution are shown below. 1 2
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Figure -1 : pGlo Plasmid. 1 Figure – 2 : GFP Structure. 6 3
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Figure – 3 : Mechanism of GFP production from pGlo plasmid. 1 B. Protein Purification : In this experiment the green fluorescent protein (GFP) was purified from an E.coli culture and the protein was analyzed using SDS polyacrylamide gel electrophoresis. 2 Electrophoresis is a process in which charged molecules are separated in an electric fired based on their mobilities. The separation of proteins depends on pH, size, and frictional resistance and it is desirable to separate the proteins only upon their size. Therefore, separation of protein based on size can be accomplished by using a detergent known as sodium dodecyl sulfate (SDS) that denatures proteins. SDS binds strongly to proteins and denatures the proteins into a linear polypeptide that is coated with SDS. The charge-to-mass ratio is the same for every
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