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.
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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. Introduction 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 fragments. The two fragments will the will join by ligation and will bind any two Bam HI sticky ends together and two Hind III ends together.
The combination of these fragments will create different circular DNA molecules. Red fluorescent proteins (RFP) are found in a sea Discosoma anemone found in the coral reefs (Arkady F et. l, 2000). It is thought that it is used in the photoprotection of the sea anemone’s symbiont microalgae’s photosystems from photoinhibition caused by high levels of incident light found near the surface of coral reefs. The protein has 225 amino acids and has a mass of 25,931 Daltons. Proteins are separated by using polyacrylamide gel electrophoresis (PAGE). Amino acids are smaller units that make up the protein. Peptide bonds join the amino acids to form polypeptide chains. These chains make up a protein and can interact with other polypeptides to form multi-subunit proteins.
The proteins are treated with the detergent sodium dodecyl sulfate (SDS) and heated. The protein tertiary and quaternary structures are denatured with the SDS and heat which makes the proteins more linear and gives it a negative charge. Proteins that contain SDS Laemmli buffer are separated on a gel. The gel is then put in a chamber filled with buffer between two electrodes. A power supply generates a voltage gradient across the gel. The proteins that are charged negatively go towards the cathode and the lager proteins moving slower than the smaller ones. The SDS coated proteins move toward the positive electrode.
Then the proteins will separate according to size. The purpose of this experiment is to introduce a method used to analyze the genetic elements of plasmid DNA, produce a recombinant DNA molecule, and identify RFP on SDS-PAGE gels. It is expected that bacteria that does not have plasmid would die.