Genetic Transformation Has Occurred Within The E. Coli Culture

This pBlu lab had for purpose to present the changes of the strain of E. coli bacteria due to new genetic information being introduced into the cell. In this experiment we are freezing and heat shocking the E. Coli bacteria that is then forced to take the plasmid DNA. The E. coli then transforms the pBLu plasmid, which carries the genes coding for two identifiable phenotypes. After following the Carolina Biological steps our lab worked well and we able to see some colonies of bacteria on the plates. The x-gal plate showed a significant amount of bacteria to confirm that the pBlu plasmid took over the E. coli strain.

The vital components and techniques of gene cloning are as follows, the DNA sequence that contains the desired gene (EZH2) is amplified by Polymerase chain reaction. PCR was established by Kary Mullis in 1985, popularly known to amplify target sequences of DNA (EZH2) to a billion fold in several hours using thermophilic polymerases (Taq) ,primers and other cofactors (Sambrook and Russell, 2001). Three crucial steps are involved which are Denaturation (at 95°), Annealing of the forward and reverse primers (55-65°) and lastly primer extension (at 72°). After amplification the desired sequence is integrated into the circular vector (pbluescript) forming the recombinant molecule. For the compatibility of the insert and vector, both were digested with (EcoR1) so the same cohesive ends are generated in both, making it easier to ligate. EcoR1 is a restriction enzyme that belongs to the type II endonuclease class which cuts within dsDNA at its recognition site “GAATTC” (Clark 2010; Sambrook and Russell, 2001).

E. coli HB101 was transformed with pGLO plasmid then grown on media containing ampicillin and/or arabinose and on medium containing neither (Brown, 2011). This is done for selection of transformed cells since not all cells are expected to take up the plasmid (Brown, 2011). We also expect roughly the same CFU on any plate(s) receiving samples from the same microcentrifuge tube, since they are getting the exact same

The purpose of this experiment is to make E.Coli competent so that it can be transformed in order to become immune to ampicillin, then we would be able to determine the transformation efficiency of the culture. We determine this by preparing 4 plates of E.coli, each labeled “LB-plasmid”, “LB+plasmid”, “LB?Amp-plasmid”, and “LB/Amp+plasmid”. This meant that either should have lacked plasmid and Ampicillin, with plasmid but lacked Ampicillin, without plasmid but with Ampicillin, or were with Ampicillin and plasmid, respectively. Then we made the bacterial cells competent by adding CaCl2 to 2 vials of the colony (one with plasmids), and incubating on ice, then heat shocking, and returning to ice. Luria Broth is then added and left to sit for 5-15

If a gene that codes for Green Fluorescent Protein transforms bacteria and GFP glows when transformation occurs, then when two micro test tubes have 250 microliters of transformation solution and places in an ice bath, then 2-4 bacteria colonies are added to each tube with a sterile loop; then a plasmid (pGLO) is added to one of the tubes, incubated in ice for 10 minutes, then heat shocked for 50 seconds at 42 degrees Celsius, then back into 9ice for two minutes; then LB nutrient broth is added to both tubes (250 microliters) and set out at room temperature for 10 minutes. Then, 100 microliters of each solution in the tube are added to four

Genetic transformation occurs when an organism’s genetic makeup is altered due to the introduction of new genetic information which is then incorporated into the organism’s genome. In this lab the pGLO plasmid is introduced into E. Coli bacteria, and incorporates the genes which code for the GFP and beta lactamase to the bacteria’s genome which as a result will be modified. To test the effects of the plasmid, bacteria treated with the plasmid were grown on separate plates, the first containing LB nutrient broth and ampicillin, another containing LB nutrient broth and arabinose and another containing LB nutrient broth, ampicillin and arabinose. Two more plates were grown, one with LB nutrient broth and ampicillin and the other with only the LB broth, using cells that did not contain the plasmid. Since the lab was about genetic transformation, the goal was to find which plate would glow. It was found that the plates that were not exposed to the plasmid did not glow, and the plates containing LB and arabinose and LB, ampicillin and arabinose did glow. The plates containing ampicillin, the antibiotic that kills E. coli did not grow whereas the remaining plates at least had some growth.

Purpose: The purpose of this experiment is to teach the students step by step on how the genetic transformation process works using pGLO. Genetic transformation is when an individual cell’s genetic material is changed by exogenous DNA.

The objective of this experiment is to conclude that the results of Griffin and Avery et al, can be duplicated in a way that will allow us to corroborate their results. The null hypothesis that this repeated experiment revolved around was that the DNA involved from the E. Coli would not undergo transformation and therefore grow a strain, which means that there would be growth on any of the plated specimens. This leads to the alternative hypothesis that only the plates that involved the DNase would not have growth as it has the heritable genetic information that would allow the transformation to occur and

The second method was to purify plasmid DNA from the E. coli culture and physcically analyze the DNA by digesting the plasmid with specific restriction endonucleases. The restriction endocucleases used were Bam HI (B), EcoRV (E), and BgIII (G). These restriction endonucleases recognize specific short DNA squences. These cut plasmids were then placed in a 1% aragrose gel to determine the band sizes. The expected phenotype for Pglo is that there will be floursenscens light produced on the LB + amp + Arab plate, where as for PFG the expected phenotype is that there will be flourcent phenotype only in LB+ Amp + IPIV. For the physical analysis Pglo will produce fragment sizes in restriction enzyme (E) 5371bp long. Enzyme (B). fragment sized 625bp, 220bp, and 4,526bp in restriction enzyme (B). Pfg will produce 2 fragments in restriction enzyme (E), one will be around 3,855bp long, and the other one will be 2,506bp long. In restriction enzyme (B) there will be only one band about 6361bp long. From restriction enzyme (G) there should be one bad around 6361bp

A restriction enzyme is an enzyme that acts as a catalyst that can cut a strand of DNA in a particular place.

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 purpose of this lab is to observe bacterial growth under various conditions including the transformation of bacteria; to understand how the process of transformation occurs.

In the following experiment, Escherichia coli were transformed using six separate agar plates. Transformation occurs when plasmid DNA is uptake into the bacterial cell. The two ways transformation is facilitated is by placing them in calcium chloride (CaCl2) and heat shock. These two methods allowed the bacterial cells to become competent or more open to the uptake of plasmid DNA. The purpose of this experiment was to transform bacterial cells with plasmid containing ampicillin resistance and lux genes. It was hypothesized that the lux genes would have created a light-emitting reaction on the bacteria while the ampicillin genes should have made the bacteria resistant to ampicillin attacks and be able to grow normally. The experiment results

coli) with a pGLO plasmid, which will cause it to take in and express the Green Fluorescent Protein (GFP) gene. This gene, which has been encoded into the pGLO plasmid, is found in jellyfish and causes them to produce the protein that makes them fluorescent green. A plasmid is a circular piece of DNA found within a prokaryotic that contains certain genes for a trait that can help the prokaryote to survive. They can also be extracted from prokaryotes and modified to express a desired gene. Bacteria naturally exchange plasmids, causing them to gain traits from the other and increase their chance of survival. To integrate plasmids into DNA, restriction enzymes cut the DNA at certain points, creating “sticky ends”. Sticky ends are areas of unmatched nucleotide bases that can be attached to corresponding nucleotide bases. Once the DNA and plasmid have been cut, the sticky ends from both are attached together so that their nucleotide bases match and are sealed with the enzyme ligase. After this, the cell can express the gene that the plasmid contains. If the E. coli bacteria successfully take in the plasmid containing the GFP gene, they will express the protein and become fluorescent green. The pGLO plasmid also contains a gene called beta-lactamase that causes resistance towards the antibiotic ampicillin. Therefore, if the E. coli bacteria successfully take in the plasmid, they will

In this experiment we are testing what is required for E. coli to successfully grow on LB (Luria Broth) plates with ampicillin and determining if any genetic transformation has occurred. By combining +pGLO LB and ampicillin we should get an ampicillin resistant gene and by using –pGLO we should create a non-genetic resistant bacteria. The pGLO plasmid has the GFP (green fluorescent protein) gene and the gene that allows the plasmid to be resistant to the antibiotic ampicillin. The most important part of this experiment is the “heat shock treatment” because the E. coli membrane becomes permeable and increases the competency of the