Transfection Lab Report

coli the bacteria that will be used to house the GFP is the competent cell. They are called competent cells because they are able to accept foreign DNA or plasmids. They are first observed in 1928 by Frederick Griffith where he proposed that bacteria are capable transferring genetic information (Havarstein, 2010). These cells such as E. coli are normally made competent by exposure to a rich calcium environment, where the mixture calcium chloride will be used in. Calcium chloride’s positive charges will cancel out the negative charge of the plasmid and E. coli’s cell wall weakening it, making it easy to pass.

The pGLO plasmid will transform the E. coli bacteria with a gene called GFP that codes for the Green Fluorescent Protein in the genetic code. GFP was discovered in the jellyfish, Aequorea victoria as a green fluorescent light emitted from the jellyfish. It was typically seen in the dark upon its activation and since then has been used in studies relating to genetic transformation. (Chalfie and Tu 1994) The majority of the studies test the many different factors that are required in the transformation of pGLO which will determine the functionality of GFP in the E.coli bacterium. The first experiment in transforming GFP and E. coli was completed in 1994 by Chalfie and was further refined the same year. The experiment proved the importance of using restriction enzymes, and DNA ligase in the process of transforming GFP to identify arabinose as the primary activator, and to identify the ampicillin

The field of biotechnology involves the concept of genetic engineering, altering the DNA/genetic material of an organism using information from a different one. The process in which bacteria can obtain this manipulated genetic information from another source is called genetic transformation. The goal of this experiment was to genetically transform Escherichia coli bacteria’s DNA by inserting the vector pGLO plasmid which codes for ampicillin resistance as well as the green fluorescent protein, GFP. For the experiment, the E. coli bacteria were separated into two groups; control and

For PEI Transfection, we added 1 ml of Trypsin to a T-25 flask of 293T cells and incubate at 37 0C for 10 minutes. Then, we supplemented 10 ml of DMEM (2.5%), FBS (15), P.S medium to the flack and finally transferred to dish and put back in CO2 incubator. After 5 days, we diluted plasmid DNA (ug) in 250 ul of 150mm NaCl in a sterile tube, and diluted 40 ul of PEI into 250 ul of 150 mm NaCl in second sterile tube. After that, incubated each separate tube for 5 minutes at RT and then mixed the DNA tube with the PEI tube. We put the tube in incubator for 2-5 minutes at RT, supplemented dropwise to cells, and put back into incubator. The important steps that was added helper virus (VSV) to cells and put it in the CO2 incubator; then, collected supernatants that contains our

coli, to express a gene that is normally causes jellyfish to be fluorescent. The plasmid that contains the gene for the green fluorescent protein (GFP) is called pGLO. The type of genetic transformation we used in the experiment was heat shock which made the membrane of E. coli fluid and allowed the pGLO to enter the cell. In order for the E. coli to express the GFP gene there needs to be arabinose presence, which is a sugar that acts as an on switch for the pGLO. Using this information, we predicted the bacteria E. coli will grow and express the GFP on the plate that consists of +pGLO, ampicillin, arabinose, and LB broth (Luria Bertani broth).

Genetic engineering is used in health treatments, agricultural applications, and environmental solutions. Genetic transformations incorporate foreign genetic material into the DNA of a different organism via a vector, which carries the genetic material. Plasmid DNA is small, round, and autonomous, due to its origin of replication. In biotechnology, plasmids carry beneficial genes, such as antibiotic resistance, and also a reporter protein, in this case, Green Fluorescent Protein

The focus of this lab was to identify which plasmid (PFG or Pglo) was inserted into the E. coli culture. In order for the E. coli to be transformed, the E.coli must first be made competent. The cells were made competent by incubating the cells in calcium (2+) and then in cold temperatures. A quick heat shot opens pores in the cell membrane and allowed plasmid DNA to enter the cells. The plasmid that was inserted into the E. coli culture had antibiotic-reistance gene and a reporter gene. A reporter gene is a gene that is easy to detect, and for this lab it could be Pglo or PFG. The cells were placed in plates that contained antibiotics to kill any bacteria that did not uptake the plasmid, this is necciary because about 1% of bacteria will be transformed. This allowed only cells that up took the DNA to

Abstract: This lab was conducted to determine the efficiency of transfection in HEK293 cells using cell culture and lipofectamine by growing the HEK293 cells out in growth medium, and then passaging them into 24 well plate at 50% confluency. After the passaging of the cells, and aspirating the media after letting the cells grow in the wells for 24 hours, then a master-mix of lipofectamine and GFP plasmid DNA was added to 500 microliters of stock DMEM, and then adding 100 microliters of the master-mix to each well, and allowing then to grow overnight. Then the next day accutase was added to dislodge the cells and placed on a side to view them under a UV microscope to find the efficiency of the transfection. The experimental had the same procedure

Western blotting is used to tell you how much protein has accumulated. It takes a protein that has been separated via gel electrophoresis and transfers it onto a blot which is then labeled with antibody and immunostained in order for the antigen bands to be visualized.

Plasmids are small double stranded circular non chromosomal DNA molecules containing their own origin of replication. Hence, they are capable of replication independent of the chromosomal DNA in bacteria. Plasmids present in one or more copies per cell, can carry extra chromosomal DNA from one cell to another cell and serve as tools to clone and manipulate genes. Plasmids used exclusively for this purpose are known as vectors. The genes of interest can be inserted into these vector plasmids creating a recombinant plasmid. Recombinant plasmids can play a significant role in gene therapy, DNA vaccination, and drug delivery [Rapley, 2000].

One use of GFP is as a component of the plasmid pGLO. pGLO is made through recombinant DNA technology and consists of the GFP gene that codes for green fluorescence, a gene (bla) that codes for the enzyme beta-lactamase which produces bacteria resistance to the beta-lactam family of antibiotics of which ampicillin is one such antibiotic and lastly AraC which binds the arabinose thereby regulating expression of GFP. GFP will not be expressed unless the carbohydrate arabinose is present. Plasmid is small pieces of DNA that is distinct from a cell's chromosomal

First, 500μl of calcium chloride was added to a DNA tube. Calcium chloride makes it easier for cells to transform. Approximately 15 E. coli colonies were then transferred to the tube. 250μl of the DNA was transferred into another tube, where 10μl of pGFP was then added. The plasmid for green fluorescent protein is what will be transformed into the E. coli’s DNA so it will express the GFP. The DNA tubes were then incubated in hot and cold water .This process is called “heat shocking” and affects the permeability of cells, allowing for new DNA to move into cells more easily. Initially the tubes were put in ice for 10 minutes, they were then put in water that was 42℃ for 90 seconds, after that they were put on ice for another 2 minutes, 250μl

Genetic engineering is the deliberate modification of the characteristics of an organism by manipulating its deoxyribonucleic acid (DNA). Bacteria possess plasmids which can be used as vectors for bacterial transformation. The plasmids can be altered to include donor DNA thus altering the physiological or physical characteristics of the bacterium. Escherichia coli have been found to be the most effective bacterium in genetic transformation and heterologous expression of human genes or proteins. It was hypothesised that Escherichia coli HB101 K12 could be transformed with a pGLO plasmid to express the genes encoded for green fluorescent protein (GFP), the bla gene for ampicillin resistance and the araC gene a regulatory protein in facilitating transcription and therefore the transformation efficiency for this bacteria could be calculated. Bacillus megaterium, E.coli B strain, Erwinia carotovora, Pseudomonas fluorescens, Staphylococcus epidermidis and Streptococcus lactis will also be subjected to transformation with the pGLO plasmid to collect transformation efficiency data for comparison against Escherichia coli HB101 K12. Escherichia coli HB101 K12 were found to have been successfully transformed with a transformation efficiency falling within the transformation protocol of 8.0 x 102 and 7.0 x 103. The incidence of Escherichia coli HB101 K12 on the LB/amp and LB/amp/ara pGLO+ plates indicated that the bacterial colonies present showed resistance to the ampicillin

The plates in this photo were mislabeled. The plate labeled LB+ amp+ Arab (+) is actually the culture grown on LB+ amp+ IPTG(+). Furthermore, the plate labeled LB+ amp+ IPTG(+) is the culture grown on LB+ amp+ Arab (+). In figure II the plate reveals that the phenotype was displayed on the medium that contained IPTG, but it is labeled with Arab. This picture was taken with gel imaging system to visualize the phenotype. The plate that labeled Arab appears darker because this plate’s cells were displaying fluorescent light. Furthermore, this reveals that the transformed plasmid contained a promoter that was induced by IPTG. A hypothesis was made at this point of the experiment; the hypothesis was that pGlo was inserted into the cells, but this hypothesis was wrong. At this point of the experiment the labels were believed to be correct, but if the knowledge of the switched labels were know, the hypothesis would have been: pFG was inserted into the E. coli culture. The way the reporter gene was determined was through the physical analysis.

The H1 proteins, as shown in figure 3, eluted in the last peak which showed a low absorbance due to a lack of reactivity. The eluted H1 protein was applied to an SDS gel in order to confirm the