Bacterial Transformation Bacteria and plasmid to produce Red Fluorescent Proteins Alejandra Lopez Biology 124L Abstract
A transformation in the literal sense of the world was witnessed in 1928 by Fredrick Griffith. A living organism had changed in physical form. The purpose of this study is to produce recombinant DNA molecules to produce bacteria that would transform into red fluorescent proteins. One plasmid was that allowed to express a red fluorescence was produced by recombining two plasmids by using molecular techniques. Agar plates labeled LB, LB/AMP, and LB/AMP/ARA containing ampicillin (AMP) and arabinose (ARA) were used to grow of the bacteria of interest and SDS-PAGE gels were utilized in identifying the fluorescent and non-fluorescent proteins. The end results illustrated that there were no signs of fluorescent proteins in the gel bands and there were red fluorescing bacteria in the LB/AMP plate that should not have been. The agars contained in all three plates was exposed to arabinose, and there is a possibility that the plates were labeled incorrectly causing the unsuccessful results.
According to the hypothesis proposed by George Beadle and Edward Tatum in 1940, the transforming principle involved one or more genes to produce enzymes needed to synthesize the polysaccharide coat. Biochemical tests revealed it to be deoxyribonucleic acid (DNA). Watson Crick Discovered the elegant structure of the DNA and the molecular genetics was born which eventually lead to our ability to produce recombinant DNA by splicing together DNA and molecules from different sources.
Bacterial Plasmids are circular closed DNA molecules that range in length from 1,000 base pairs to more than 200 Kb. They behave as independently replicating genetic units inherited of the Bacterial chromosome. They depend on their host cells for replication, gene expression and transmission, but they also carry genes encoding enzymes that are conditionally advantageous to their host. Their small size allows for the plasmids to be used as vehicles, or vectors to clone DNA of interest. With plasmid vectors, genes can be cut from human, animal, or plant DNA and placed inside bacteria via transformation. The gene insertion can usually provide the organism with a new trait (eg.pest or antibiotic resistance). For example, in humans, a gene used for the production of insulin has been cloned into a plasmid and transformed into bacteria. These transformed bacteria, under the right conditions, will produce authentic human insulin to treat diabetic patients (Crameri, et al., 1996).
The plasmids pKAN-R and pARA are cut by restriction enzymes Bam HI and Hind III. The pKAN-R plasmid has kanamycin resistant gene that encodes a phosphotransferase enzyme that will destroy the antibiotic effect. The bacteria that carries the pKAN-R plasmid becomes resistant and will carry the rfp gene that encodes a red fluorescent protein. PARA carries the ampicillin resistance that will encode the protein beta lactamase that can make them capable of reproducing even in the presence of ampicillin. pARA also carries the AraC gene that can encode the transcriptional regulation protein. It binds to a region of DNA called the PBAD promoter that controls ant RNA polymerase transcription of genes. If arabinose is present it will bind the AraC protein that will cause Arac to let go of the PBAD promoter. Digestion of pKAN-R will produce two fragments and digestion of pARA will also produce two...
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