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Pablo Transformation Lab Report

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Mawjuda Abdulrashid Dr. Jesus Segovia Cell Biology Lab 3411L March 23, 2015 Transformation of E.coli with Plasmid DNA Abstract Transformation is the process by which DNA is introduced into bacteria. And then the bacteria can replicate and express that specific DNA. The purpose of the study was to study the theory of transformation and to develop an understanding of bacterial transformation by plasmid DNA. X-gal is a special sugar for the beta galacosidase enzyme and if we place this particular sugar into a bacteria, a very interesting thing will happen. “The ‘X’ in X-gal is a colorless pigment when attached to the galactose sugar but turns blue when cleaved by beta galaxtosidase” (Covert, 2011). Therefore, we observed blue colonies in our plate. There were approximately about 145 colonies present in plate A, 132 colonies in plate B, and no colonies in plate C. Our second data supports my hypothesis. There was a negative correlation between the fragment length and the distance migrated. The equation that we obtained was -0.0035x+51.319 and the r squared value was 0.8347. We grew E.coli bacteria in three different plates and observed whether they grew blue, white, or no colonies. This particular bacteria is not able to grow on ampicillin. My hypothesis is that cells exposed to LB (nutrient mixture) with ampicillin will …show more content…

Restriction enzyme basically are enzymes that cut DNA somewhere along its sugar phosphate backbone (Cheng 2015). They are naturally found in bacteria. Bacteria can be infected with viruses and as a way of protecting themselves against viral infections, what a lot of bacteria can do is that they can make these restriction enzymes. And the purpose of making them is to chop up any viral DNA that tries to infect the bacterial cell. The most important thing about restriction enzymes is that they cut the DNA at a particular and specific location, not random sequences (Nassan,

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chinesis. A construct of R751::Tn4351 (the physical map of R751::Tn4351 and restriction sites are shown in fig. 7) was selected for introduction into F. chinesis to discover if the introduction and insertion of the vector R751 and the transposition of T4351 into the F. chinesis chromosome by a triparental mating occurred. One parent was E. coli GJ342 which carried a helper plasmid, the second parent was E. coli HB101 which contained R751::Tn4351 and the third parent was the F. chinesis target strain. 189 colonies were isolated on LB agar plates which in passage in fresh media were able to grow in 200µgml-1 erythromycin.

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How could a single nucleotide change on the plasmid alter the fragment pattern observed in the gel? (3 points) When a single nucleotide is change on the on the plasmid, it will make the restriction enzyme not to recognize the fragment pattern and also all the numbers at each site will be altered or this action could make the fragment not to be incised at the expected site, which could make a larger fragment to travels a short interval on the gel with less fragments than expected. It could also make it to be incised at more places than that originally expected and the result will be increased, smaller fragments that move further across the gel. There may also be no change in the banding pattern.

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The other mediums were lacking all the necessary components for the activation of the GFP protein. The non-transformed E. Coli will be in the LB and LB + ARA plates since there is no ampicillin to kill

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First, label one micro centrifuge tube +pGLO and another –pGLO. Using a sterile transfer pipet, transfer 250µl of competent cells (E. coli + CaCl2) into each tube and place them in crushed ice. Examine the pGLO plasmid DNA solution with the UV light and note your observations. Pipet 10µl of pGLO plasmid into the +pGLO tube and mix, close and return it to the ice rack. Do NOT add plasmid DNA to the –pGLO tube.

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The plate without the plasmid that contained ampicillin, and the LB broth did not grow because ampicillin is an antibiotic that impeded the growth of bacteria because it killed off. There was no glow because it didn’t contain the green fluorescent protein gene or any other of the components to make it glow. The plate that contained the +pGLO, the ampicillin, and the LB broth was able to grow, because the plasmid provided the ampicillin resistance gene and the LB broth to feed the bacteria, however it wasn't able to glow because no arabinose was present to activate the GFP gene. Another plate that was able to grow was the one without the plasmid and just the LB broth. Finally the plate with all the components, +pGLO, arabinose, ampicillin, and broth was able to both grow and glow because the arabinose (sugar) turned the GFP gene on, the resistance to ampicillin gene was present and that let the bacteria survive and feed on the LB broth which let the culture

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In this lab, the goal was to transform bacteria with genes that included fluorescence as well as antibiotic resistance that were taken from a jellyfish. Transformation is transferring a gene from one organism to another. Certain precautions had to be made before doing this lab since every step had to be done very quickly to prevent too much contamination. The first step in starting the transformation is to add the transformation solution into the +pGLO and -pGLO test tubes. After this is done, you put both tubes in ice and then put bacteria in both tubes.

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The hypothesis for this experiment was that transformed bacterial cells would grow on ampicillin plates and glow green when exposed to UV light. The rationale for this hypothesis was because the plasmid would code for ampicillin resistance and a green fluorescent protein, we would have the outcome explained in the hypothesis. 4. Our predictions were that for the standard protocol plates with agar and ampicillin, there would be growth and the colonies would glow under UV light. On the modified protocol on plates with agar and ampicillin where we changed the time of the heat shock, we expected less growth but the colonies would still glow.

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In addition, the bacteria need arabinose that help activate the protein that allows to glow green in the transformed bacteria cells. To let

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B-galactosidase breaks down the disaccharide lactose into simple sugars glucose and galactose. However, glucose is a colorless compound hence it has to be substituted with a compound that is detectable by a visible color change. Hence,

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In the presence of ampicillin, E. Coli growth will not occur. Therefore, in this experiment, bacterial growth on the plate indicates the uptake of the ampicillin resistant gene into the genetic code of E. Coli. If pGLO, LB broth, ampicillin resistance, and arabinose sugar are added, then the bacteria will glow. In one of the four plates that include all of those component, fluorescence will be observed. The purpose of this experiment is to demonstrate the genetic transformation that occurs in the E. Coli bacteria.

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Our hypothesis was that if the plate contains only the LB broth the E. coli bacteria would have no antibiotic resistance and would not glow. If the plate contains just LB broth and ampicillin then the E. coli bacteria will have antibiotic resistance only if the plasmid is present. If the plate contains LB broth, ampicillin and arabinose then the E. coli bacteria will glow fluorescent under a UV light and it will have antibiotic resistance. Similar to our expectations our results suggested that our hypothesis was correct. This is due to the fact that n order for the E coli.

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Hypothesis The transformed E.Coli with the ampicillin resistance gene will be able to grow in the ampicillin plates and it would have a green glowing color.

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