In the “Transformation with Green Fluorescent Protein (GFP)” lab there were two samples of bacteria. One of which had been exposed to the GFP (+DNA) and one that was not (-DNA). The two DNA samples were subjected to a heat shock process. Both tubes incubate on ice for 10 minutes and are then are placed in a 42* C water bath for 90 seconds. Immediately after that transfer the tubes back to the ice for 2 minutes after that the tubes are put into a 37* C water bath. The heat shock process facilitates the entry of the plasmid into the bacteria. The plasmid carries the GFP and the antibiotic resistance genes. The samples were placed on one of four petri dishes labeled; -DNA, -DNA/AMP, +DNA/AMP, and +DNA/+AMP/+IPTG. AMP or ampicillin is an antibiotic …show more content…
The growth on the -DNA petri dish was more than 300 colonies of bacteria, the -DNA/+AMP had no growth, the +DNA/+AMP had a total of 3 colonies, and the +DNA/+AMP/+IPTG had 2 fluorescent green colonies. The results obtained were expected. The -DNA dish was expected to have a lot of growth due to the lack of the AMP. The -DNA/+AMP dish was expected to have no growth due to the -DNA not being exposed to the plasmid so it could not have developed a defence against the AMP. The +DNA/+AMP was expected to have some growth due to +DNA being transformed with the antibiotic resistance genes. The +DNA/+AMP/+IPTG was expected to have very little growth due to both genes, GFP and the antibiotic resistance genes, having to be transformed. One experimental error that could have caused the results to be unexpected is the amount of time the petri dishes were incubated for. Some groups were not able to complete the full 30 minutes. The technology demonstrated in the lab is used in genetic engineering. Scientists use this in gene therapy.Gene therapy is used to change a broken gene. Many organisms are subjected to gene therapy including humans. The inserted gene comes from a donor with the normal gene. The gene is inserted to a plasmid and then into the patient. The plasmid replicates along with normal gene. The patient could have a cancer causing gene. Gene therapy would be able to insert a normal gene and prevent the
Once the recombinant plasmid was obtained, it was then inserted into E. coli cells through transformation. From a successful transformation, we expected the bacterial cells to translate the inserted EGFP sequence into its protein form. The bacteria cultures were plated on petri dishes containing growth supplement, Luria Broth (LB), an antibiotic: Kanamycin, and IPTG which induced the fluorescence property within successfully transformed bacterial colonies. Different variants of the petri dishes were also included as control and unknown.
pBK-CMV is a plasmid vector 4518 in size, it also contains a multiple coding site (polylinker) that has recognition sequences for many restriction endonucleases. cDNA molecule CHI-1, which is 600bp, has been previously inserted. pUC19 is a cloning vector developed by….. in …….at….(REF). This vector is 2686bp in size and contains a 54 base pair (bp) polylinker containing 13 specific restriction sites, Xba1 and EcoR1 inclusive. It makes a good cloning vector as it is small in size, this makes it easier to be taken up by its host during transformation and allows for a faster replication time (Green, 2015). It contains an origin of replication pMB1 which is essential to be able to replicate. pMB1 has a high copy number allowing for multiple copies to be made (REF hcn pmb1). The pUC19 plasmid vector contains an ampicillin resistance gene, the host containing this plasmid will survive in the presence of ampicillin allowing for the selection of transformed host bacteria. The polylinker of pUC19 is contained within a lacz’ gene allowing us to distinguish between recombinant pUC19 and non-recombinant pUC19 through a process call insertional inactivation (Green, 2015).
al. (1994) explain that a complementary DNA for GFP produces a fluorescent product when expressed in E. coli cells as the expression of GFP can be used to monitor gene expression and protein localization in living things. In this experiment, the heat shock method will be used to deliver a vector (plasmid) of GFP to transform and grow E. coli bacteria. Four plates containing Luria Bertani (LB) broth and either –pGLO or +pGLO will have E. coli bacteria added to it. The plate containing –pGLO (no pGLO) and LB will show growth as ampicillin will be present killing bacteria but no glowing because no arabinose will be present for glowing to be activated, the same result will be seen in the plate containing +pGLO, LB and ampicillin.
Germline gene therapy is where the correct "good" gene is inserted into the germline in place of the defective "bad" gene, and when reproduction occurs the gene will be passed on to the progeny. Inserting the "good" gene into the very early embryo sta ges of development allows for both germline and somatic cells to be corrected. Government has limited the research to only somatic cell gene therapy such as performed in Cystic Fibrosis research.
...et light. If the LAA plate glows green under exposure to ultraviolet light, then we can conclude that our unknown insert piece of DNA would be the kan gene. If it does not glow green under exposure to ultraviolet light, then then we streak the colony from our LAA plate onto the LAC plate using a sterile glass spreader. When the LAC plate is dray, we place it upside down in the microfuge rack so that it can be incubated at 37 ºC. Incubation at 37 ºC will allow the transformed bacterial cells to grow. If we see bacterial growth on the LA plate containing chloramphenicol, we can conclude that our unknown insert piece of DNA would be the cat gene, since the cat gene is resistant to chloramphenicol. Afterwards, we then grab the microfuge tube labeled NP and repeat the aforementioned steps shown above pertaining to the LA plates. This would be considered our control.
Human gene therapy is a method used in the medical field that treats diseases at a molecular level, by solving the source of the problem; our genes. Today, diseases and disorders are commonly treated by solving the symptoms, the surface of the problem. Many disorders and diseases are caused by defective proteins and within those defective proteins are damaged and defective genes. These defective genes can be treated through gene therapy. Gene therapy is not new and has been developed and improved by researchers for the past couple years. Being an experimental technique, gene therapy also has its pros and cons, but so far is showing positive and rising success rates.
Over 40 years ago, two men by the names of James Watson and Francis Crick discovered deoxyribonucleic acid, or DNA. DNA is hereditary material in humans and almost all other organisms (What is DNA?). From this finding, gene therapy evolved. Today, researchers are able to isolate certain specific genes, repair them, and use them to help cure diseases such as cystic fibrosis and hemophilia. However, as great as this sounds, there are numerous ethical and scientific issues that will arise because of religion and safety.
Protein Concentration; Utilizing Spectrophotometer to analyze commercially available food products to Justify FDA food Labels
Gene therapy is a relatively new concept owing mainly to our current knowledge of the human body and the relatively modern understanding of genetic coding and process. We now are able to better identify and understand the genetic causes of human ailments, and are just beginning to understand how to fix, replace, or eradicate the chromosomal basis for these issues; this is the concept of gene therapy. However logistically dealing with the small structure of genes, chromosomes, and DNA is not as easy as repairing a cracked wall or damaged water pipe, we are dealing with complex and microscopic materials that ordinary tools cannot deal with. Manufacturing such tools to deliver corrective DNA into affected cells within the body is just one of the obstacles that scientists and researchers are facing.
Although the highly technical aspects of human gene therapy are somewhat complex, the basic concept is very straight forward. The goal of gene therapy is to correct mistakes that have occurred within the genetic material, or DNA, of the living cell. In very simple terms, DNA is often thought of as the "language" of the biological functioning of organisms. This language is organized by letters (nucleotide pairs), words (codons), sentences (genes), and books (genomes). Before being able to repair the damaged or defective genetic material, the location of the gene or genes causing the dysfunction in the individual must be determined.
Genetic engineering is now being used to create new medicines and therapies for many disorders and diseases, and also to improve agricultural plants and animals to produce bigger yields or enhanced nutrient composition and food quality. In Gene therapy, copies of healthy human genes produced in bacteria can be inserted into human cells with defective or missing genes, to fix the problem. Gene therapy is promising because it can use to treat genetic
Antibiotic resistance is bacteria’s loss of susceptibility to the bactericidal or growth-inhibiting properties of an antibiotics. When a resistant strain of bacteria is the dominant strain in an infection, the infection may be untreatable and deadly he primary mechanisms of bacterial gene transfer are transduction and conjugation. Transduction occurs when a bacterial virus, called a bacteriophage, detaches from one bacterial cell, carrying with it some of that bacterium’s genome, and then infects another cell. When the bacteriophage inserts its genetic content into the genome of the next bacterium, the previous bacterium’s DNA also is incorporated into the genome. Conjugation occurs when two bacteria come into physical contact with each other and a plasmid, sometimes carry...
On the other hand, cells that have resistance from the start or acquire it later may survive. At the same time, when antibiotics attack disease-causing bacteria, they also attack benign bacteria. This process eliminates drug-susceptible bacteria and favors bacteria that are resistant. Two things happen, populations of non-resistant and harmless bacteria are diminished, and because of the reduction of competition from these harmless and/or susceptible bacteria, resistant forms of disease-causing bacteria proliferate. As the resistant forms of the bacteria proliferate, there is more opportunity for genetic or chromosomal mutation (spontaneous DNA mutation (1)) or transformation, that comes about either through a form of microbial sex (1) or through the transference of plasmids, small circles of DNA (1), which allow bacteria to interchange genes with ease.
Gene therapy is a provisional technique that is the insertion of normal genes into the cells where there is a missing or miscoded gene to fix a genetic disorder. In the 1960s and early 1970s,
Moses, R. E., & Summers, W. C. (Eds.). (1988). DNA replication and mutagenesis. Washington, DC, WA: American Society for Microbiology.