E. Coli And Plasmid Transformation

The instructor split the class into two separate groups one with the plasmid lux and the other with the plasmid pUC18. Group two was assigned to test the lux plasmid. The, Eppendorf, tubes were labeled “C” for the control plasmid DNA and “lux” for the plasmid lux DNA. The two tubes were then placed into the ice bath. Using a sterile micropipette 5 uL of the lux plasmid was added to the tubes labeled “lux” or 5 uL of the control plasmid was added to the tubes labeled “C” for the control plasmid DNA. Eppendorf tubes had 70 uL of the competent cells added to them with a different transfer pipet. All the tubes were then stored in the ice bath for about fifteen minutes. Another test tube was labeled “NP”, which stands for “No Plasmid”, and 35 uL of competent cells was added to each of the test tubes labeled “NP” during the fifteen minutes. Once the fifteen minutes are up, all three tubes were placed into a preheated water bath at 37 °C for about five minutes. To both the lux

DNA. The isolated plasmids are ran on 40 ml 1% agarose gel at 110 volts with 1.2 μl 6X track

After the substrate solution was added, five drops of the enzyme were quickly placed in tubes 3, 4 and 5. There were no drops of enzyme added in tubes 1 and 2 and in tube 6 ten drops were added. Once the enzyme solution has been added the tubes were then left to incubate for ten minutes and after five drops of DNSA solution were added to tubes 1 to 6. The tubes were then placed in a hot block at 80-90oC for five minutes. They were then taken out after the five minute period and using a 5 ml pipette, 5 ml of distilled water were added to the 6 tubes and mixed by inversion. Once everything was complete the 6 tubes were then taken to the Milton Roy Company Spectronic 21 and the absorbance of each tube was tested.

To do a transformation one micro test tube was labeled +pGLO and the other was labeled –pGLO, using a transfer pipette 250 µL of transformation solution was added to each tube in the foam rack. The tubes were then placed on ice for three minutes. During these three minutes, a sterile loop was used to pick up a single colony of E. Coli from the starter plate by gently running the loop over the agar. This loop was then inserted into the +pGLO tube and the loop was spun until the entire colony dispersed. Using a different sterile loop, the same procedure was used for the –pGLO tube. After both tubes had their own colony of E. Coli, they were placed on ice for another three minutes. DNA plasmid was added to the +pGLO tube by taking a new sterile loop and immersing it into the stock tube creating a film across the loop then inserting

Griffin was a scientist that showed that there was a non-living component to heritability between organisms, although he had not yet discovered what exactly was the heritable component. However, during this time period, it was a common belief that the heritable material was contained within the protein of the organism, not the DNA that is now held in the truth. In the Avery and company experiment, they expanded Griffin research to pinpoint the exact location of the heritable information by breaking down cell components until the final product allowing transformation was

After we prepared the tubes, we added PPO extract and noted the change in color after 2 minutes had passed. In the next part of the lab experiment, we created serial dilutions using freshly cleaners test tubes. We created 1in 5, 1 in 10, 1 in 20, and 1 in 40 dilutions. The c1v1=c2v2 formula was used to calculate V1 prior to starting. Tube A was the only tube that had more than 5mL as its final volume.

In the 1960s Stuart Linn and Werner Arber found both modification enzyme and restriction nucleases in extracts of E. coli strain B. This explained why sometimes when E. coli was transfected

Through performing bacterial transformation using the pB325 plasmid, the expected result was to see growth on all plates with +DNA (four plates) and two -DNA plates, the plates with no amp added. This was expected since +DNA E. coli were protected from amp and could grow on normal plates. The -DNA plates with amp were expected to have no growth as the E. coli were not protected against amp. The result of this experiment yielded a lawn of bacteria on all LB plates while none of the LB+amp plates yielded any growth. These results suggest that the experiment did not work as intended. This is because transforming the E. coli with pB325 plasmid was expected to offer those cells protection against amp. Thus, it can be hypothesized

The recommendations before starting the lab are to conduct a practice experiment with inserting the DNA with the micropipette. This decreases the chance of rupturing within the gel, and if this is to happen inaccurate results will be displayed. To begin the experiment, insert the gel into the electric chamber filled with THE solution so that it covers the agarose gel. Once the agarose gel is in place, take the Missouri DNA and extract from the sample tube with the micropipette and insert it into one of the wells. Repeat this process with each tube, but after inserting a DNA replace the disposable plastic tube with a new tube, so no inaccurate results will occur. After all six wells are filled, enclose the chamber and initiate the chamber with

Then 30 µl of GelRed was added to the agarose-TBE solution and then mixed thoroughly. The gel was then poured onto a tray with a comb inserted to create the wells and was left to set for about 30 minutes.

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.

Transformation is used in several areas, such as DNA cloning (make multiple copies of DNA) and to produce large amounts of human proteins (insulin). Transformation is the change in genotype and phenotype due to the assimilation of external DNA by a cell (Urry et al. 2016). When naked DNA is incorporated into bacteria’s genetic material, this is when transformation occurs in bacteria. The success rate of genetic transformation is more significant and faster in bacteria, such as E Coli due to the single-celled trait of the bacteria. When these types of experiments are usually performed, a plasmid DNA is used. A plasmid is a small circular DNA molecule that carries accessory genes separate from those of a bacterial chromosome; in DNA

This solution prevents endonucleases and proteins from being released during the lysis. With out this solution the quality of the DNA would be adversely affected. The time had to be carefully watched because prolonged exposure to the protease would cause some of the plasmid DNA to be nicked. 350µl of Neutralization Solution was added after five minutes to prevent nicking of the plasmid DNA. The bacterial lysate was then centrifuged at a maximum speed of 14,000-x g for ten minutes. About 850µl of cleared lysate is transferred into a prepared spin column, a spin column inserted into a 2ml collection tube. Only the supernatant was transferred into the column, the white precipitate was discarded. The supernatant was then centrifuged for one minute, and then put into a new collection tube with a 750µl diluted Column Wash Solution. The supernatant was centrifuged again for one minute; this wash procedure was then repeated with 250µl of the Column Wash Solution and centrifuged for two minutes. The spin column was put into a new, sterile 1.5ml microcentrifuge tube. The plasmid DNA was carefully eluted by adding

The first part of the experiment was to isolate the human genomic DNA from cheek cells. The cheek swabbed and cut off the end and inserted it into a microfuge tube. 500 μL of PBS was added to the tube. Then, the tube was shacked and the swab removed then centrifuged for 1 minute at 8,000 RPM. After it was centrifuged, a supernatant was formed with was removed. 300 μL of ACL solution was added to the pellet as well as 20 μL of protease stock solution. This was incubated at 50 ˚C and the temperature rose up to 80˚ C for 10 minutes and centrifuge the tube 30 seconds to remove drops from the lid. The tube was left to cool down then proceeded to be centrifuged and vortexed. Then, 250 μL of supernatant was pipetted into a spin column and an addition