Ampicillin On Bacteria With Plasmid

Transformation is the transfers of virulence from one cell to another, through the transferring of genetic material. It was originally postulated in 1928 through the works of Federick Griffith, a British microbiologist. Griffith observed that the mutant form, non-virulent form, of the bacteria Streptococcus Pnumoniae could be transformed into the normal, virulent form, when injected into mice along with heat killed normal forms. He concluded that somehow the information the dead virulent form had transformed the mutant form into a virulent form.

Transformation is the directed modification of a genome by the external application of DNA from a cell of different genotype (Griffiths and Miller).Bacterial transformation is the easiest type of transformation to create and study due to the single cellularity of bacteria and its

Bacteria are the natural world 's unsung heroes. They receive bad rep due to the disease causing individuals like tuberculosis but the vast majority of bacteria on Earth are harmless if not beneficial to both the environment and humans. Take for example E. Coli a well known bacteria that lives within our intestines. This particular bacteria makes vitamins that we need in order to stay healthy. In this experiment we analyzed the changes seen in bacteria when adding to differing DNA plasmids; pUC18 and lux. To arrive to the most accurate results possible we had to have the E. Coli bacteria made permeable to the plasmids. So it was prepared with calcium chloride. One group of bacteria was given the pUC18 plasmid, and the other group was given the lux as well as the pUC18 plasmids. We then proceeded to incubate them in different containers. Some had ampicillin where as some had none. This was done in order to observe the effect of pUC18, and lux growth patterns. A control with neither plasmid was used to keep a basis for our experiments. Bacteria that had transformed and taken in the lux plasmid without ampicilin were able to illuminate, and only bacteria that gained the pUC18 were capable of surviving with ampicillin. The results observed reveal how foreign DNA can be adapted to fit into another cell 's DNA even incorporated to be an optimal part of the cell. This process makes it a very viable manner of being able to mass replicate advantageous genes, that would

Our results show no growth whatsoever in those plates containing Ampicillin; this indicates that we encounter an error during our experiment. The agar plate's outcomes and bioluminescent response done by the bacterium that had the plasmid, it can be presumed due to scientific analysis that Escherichia coli is impervious to ampicillin and the plasmid combines itself with the DNA of Escherichia coli according to other experiments and based on science itself. We can predict that the impact of the bioluminescence in the cells of the microorganisms that is infested unmistakably gives affirmation that the plasmid infuses with Escherichia coli's DNA, guarding the cells that changed from dying, viably creating a gainful situation for the bacterial organisms. Since Escherichia coli is a negative prokaryotic call, it is within the phospholipid bilayer and on top of this is a peptide glycan

The E. coli that was not exposed to the pGLO plasmid and was in contact with LB broth and ampicillin (amp) had no bacterial growth. There is no growth in this Petri dish because amp is a bacterial antibiotic.

The transformed bacteria showed growth despite the presence of ampicillin (Table 1), whereas the control plate with ampicillin did not show any growth, and the control plate with only LB agar showed the formation of a lawn of bacteria (Table 2). The transformed bacteria on the plate with LB, ampicillin and arabinose differed from the transformed plate without arabinose in that they glowed green under UV light. The bacteria without arabinose maintained an unaltered appearance under UV light. The transformation efficiency for the transformed bacteria was 5.2 × 104 transformants per microgram of DNA.

The 2nd method consisted of purification of the plasmid DNA provided (bacterial culture). 1.5 ml of 3 bacterial culture was added to 3 different centrifuge tubes and then centrifuged for 1 minute at about 8000 xg. The supernatant material from all 3 tubes was then discarded into 3% Virkon solution and the tubes were placed back in the rack. Then 250 µl of P1 Lysis Buffer was added to the tubes and vortexed followed by an addition of the same amount of Lysis Buffer P2 and mixed gently by inverting the tubes 6-7 times, the tubes were then left to incubate for 5 minutes at room temperature. 300 µl of Neutralisation Buffer P3 was added and then mixed thoroughly by inverting the tubes. All the tubes were now centrifuged at 11,000 xg for 5 minutes taking into consideration that the centrifuge machine was balanced when used to avoid any incomplete mixture. The tubes were then removed and 750 µl was carefully added to 1.5 ml spin columns as to not disturb the white residue (Na), the tubes were then again subject to centrifuge for 1 minute at 11,000 xg and the flow through discarded after the run. 500 µl of Wash Buffer PW1 was added and centrifuged for a minute (at the same xg), after

When adding the plasmid DNA to the +pGLO tube, the scientists tried to use the loop to get 10μ to get a film of plasmid across the circle; however, they could not. This could have affected the resistance to ampicillin and the visible green glow of the bacteria. Some contamination that might have occur is when the scientist forgot to close the lid to one of the plates or did not close the bag containing the sterile loops, which could have resulted in bring other bacteria into the

Plasmid DNA is negative and bacterium’s cell wall is negatively charged. Thus, as DNA approaches the cell wall it is repelled. The calcium chloride neutralizes the charge of the bacterium and DNA therefore allowing the Bacterium cell wall to be in close proximity to a plasmid DNA. The bacterium undergoes heat shock which is technique of place a bacterium in extreme temperature with wide range. In the lab the bacteria was place in an

Plasmids are the extra-chromosomal DNA in which most bacteria contain in addition to their bacterial chromosome. Plasmids can be used as vectors to transfer DNA into a host bacterium. A nonpathogenic strain of Escherichia coli (E. coli) HB101;K-12, is the host bacterium used to take up the plasmid with the imported gene to be observed. pGLO is a recombinant plasmid that consists of a gene that codes for Green Fluorescent Protein (GFP) and also contains a gene, beta-lactam antibiotic (bla), that expresses antibiotic resistance. GFP is a protein that is produced by the jellyfish Aequoria victoria and has the ability to fluoresce green when exposed to ultraviolet (UV) light. The gene for GFP can be turned on in the transformed bacterial cell in the presence of the sugar, arabinose. The bacteria that contain the beta-lactam antibiotic have the ability to create beta-lactamase, which is an enzyme that hydrolyzes the beta-lactam rings of antibiotics which penicillin derivatives (among a broad range of others) contain, deeming them incompetent. Beta-lactam antibiotics inhibit the peptidoglycan layer of the bacterial cell wall and because E.coli is a gram-negative bacteria, its peptidoglycan layer is easily susceptible to inhibition.

Ampicillin is a beta-lactam antibiotic which can be used to treat a large number of infections. For example, Escherichia coli (E. coli) bacteria is terminated by this specific antibiotic. Ampicillin interferes with the formation of bacterial cell walls and thus kills newly dividing cells that must form new cell walls. Plasmids contain genes that create antibiotic resistance to their host cell. The pGlo plasmid contains an Ampicillin resistance gene. Therefore, bacteria that take up the plasmid and transform become resistant to Ampicillin. To carry out this experiment, my colleagues and I took four petri dishes containing the bacterial host cells (E. coli) on each and the Ampicillin on two of those four. Through a tedious process, we added the pGlo plasmid to one petri dish of just the host cell and one petri dish of the host cell and the Ampicillin antibiotic. When examining the results of our experiment, we noticed that both petri dishes containing zero Ampicillin entirely submerged in a lawn of the Escherichia coli bacteria, the petri dish containing no plasmid but some Ampicillin displayed zero growth, and the petri dish containing the plasmid and Ampicillin showed individual colonies of the E. coli bacteria glowing under a UV light. The reason this occurred is because the petri dishes that showed a lawn of bacteria contained no Ampicillin so there was no antibiotic to kill the bacteria off. The petri

Genetic Transformation in its simplest definition is the alteration of a cell when foreign material such as DNA is introduced through its cell wall, altering its genetic makeup. Genetic Transformation is significant in modern society because of its effect on the agriculture industry and crop production throughout the world. Scientists and farmers everywhere are constantly looking for ways to increase yield and quality of produce, which they have found possible with Genetic Transformation. The agriculture industry is just one common example of an industry that utilizes Genetic Transformation in order to achieve their goals and improve productivity. This process occurs through multiple techniques known as projectile bombardment,

Then added 200 ul of P1 Buffer (Red) to the tube and re-suspend pellet completely. We then centrifuged at 11 rpm for 1 min and discarded the supernatant. Then we added 200 ul of P2 Buffer (Green) and mix by inverting the tube 2 - 4 times. Cells are completely lysed when the solution appears clear, purple, and viscous. (Proceed to the next step within 1-2 minutes) We then centrifuged at 11 rpm for 1 min and discarded the supernatant. We added 400 ul of P3 Buffer (Yellow) and mix gently but thoroughly. The sample will turn yellow when the neutralization is complete then incubate the lysate at room temperature for 1-2 minutes. We then centrifuged at 11 rpm for 2 min and transferred the supernatant to a Zymo-Spin column. Once transferred we spun for 30 seconds at 11,00 rpm and then spun with 200 ul of Endo-wash for 30 seconds then 400 ul of plasmid wash buffer for a minute. After that we spun our column empty for an additional minute to remove an excess alcohol. We then transferred out column to a clean centrifuge tube and added 30 ul of Elution Buffer to the column and incubated at room temperature for a minute and centrifuged for 30 seconds. The result was our plasmid DNA.

Genetic transformation is a process that occurs when the DNA of a cell is altered by the introduction of genetic material from a different source, like from another cell or a virus. This process was discovered in 1928 by Fred Griffith while he was performing an experiment where he injected different combinations of live and dead strains of Pneumococcus bacteria that was either virulent or non virulent into mice. When only the dead virulent strain was injected, the mice were unaffected, but when both the dead virulent and the live non virulent strains were injected, the mice died. This occurred because an instructional agent passed between the living and dead bacteria, causing the DNA of the living non virulent strain to be transformed and produce

In this experimental part of the lab, two bacterial suspensions were made by adding 250µl of a transformation solution of calcium chloride, as well as adding and completely dispersing two large colonies of E. coli of approximately 2 mm in diameter, that were collected from the starter plate. Each suspension was labeled, one as +pGLO, and the other as –pGLO. 10µl of p-GLO plasmid was added to the +p-GLO suspension. Both suspensions were incubated for a period of 10 minutes on ice.