Ampicillin On Bacteria With Plasmid

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.

Next heat shocks the tubes for 50 seconds, followed by icing for 10 more minutes. The heat shock increases the permeability of the cell membrane to DNA. Then add 250 milliliters of LB and incubate for 20 minutes. The 20 minute incubation following the addition of LB broth allows the cells to grow and express the ampicillin resistance protein, beta-lactamase, so that the transformed cells survive the subsequent ampicillin selection plates. Plate 100 microliters the + tubes evenly on two plates; 1 of LB and Amp, and one of LB, AMP, and ARA. Plate 100 microliters of the – tubes evenly on two plates; 1 of LB and AMP, and one on LB only.

Control Plates After Incubation shows whether there was growth of E. coli without the presence of the pGLO plasmid after exposure to ampicillin.

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 data that my group obtained during this experiment was almost identical to my hypothesis.  On the -DNA LB plate there was bacteria growth because there was no ampicillin to keep the bacteria from reproducing.  On the -DNA LB/amp plate there was no growth because of the ampicillin that kept the bacteria from growing into colonies. The +DNA LB/amp plate had about 234 colonies of bacteria. Not all of the bacteria on the plate were able to grow into colonies but some did because they interacted/picked up the plasmid. Finally, on the +DNA LB/amp/ara transformation plate most, if not all of the bacteria were transformed and, additionally there was also arabinose sugar which activated the araC gene that codes for the green glowing protein which made the bacteria glow up when we shined the UV-light

This plate introduced the bacteria to the plasmid, but not all of them accepted the plasmid, which resulted in only some of the bacteria to undergo transformation. Since we don’t have the ampicillin environment that only selected the transformed bacteria, there remains many genetically different bacterial cells, which will form a lawn. A lawn is created when there are multiple bacterial cells present on the same plate. The reason why the lawn doesn’t glow is because the cells discarded their plasmid since it didn’t provide any advantage in an environment with no ampicillin. The role of the Luria Broth was to provide “food” for bacterial

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 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

There was a mix up with placement of the Plasmid containing Bacteria (experiment’s culture) and control bacteria. The experiment’s culture was place in the jar with no ampicillin. As a result, bacteria with plasmid grew as well as bacteria without plasmid. However, it is possible to identify the transformed culture because of the blue marker.

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.

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.

The plasmid can be inferred to have the gene resistant to the antibiotic; thus, giving the transformed cells the ability to live while in the presence of ampicillin.

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.

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

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