Pglo Lab Report

The plasmid used is called the pGLO plasmid, which has been genetically engineered. The pGLO plasmid carries the GFP gene. When GFP is produced, the transformed bacterial colonies will glow bright green under UV light. Green fluorescent protein (GFP) is the trait we are primarily looking for in this experiment. For growth, the positive control was the -pGLO LB dish because it had nothing to inhibit its growth, and the negative control was the -pGLO LB/ampicillin dish, because it did not have the genes for Beta-Lactamase to protect it from ampicillin, so it could not grow.

The color of the bacteria was a whitish color and the colony size is similar both before and after the transformation. The best way to do it is to compare the control of the experimental plates. Cells that were typically not treated with the plasmid could not grow on ampicillin, although cells that were treated with the plasmid can grow on the LB/AMP plate. The plasmid would have to confer resistance to ampicillin. Moving on, the GFP gene is what is glowing in the plate because it was activated by the sugar arabinose. The sugar arabinose and the plasmid DNA are also needed to be present because that is what initially turns on the GFP gene which makes the bacteria glow. Organisms can also turn on and off particular genes for camouflage reasons. An organism would benefit from turning on and off certain

70µL of competent E.coli are added to both test tubes; pUC18 and Lux (Alberte et al., 2012). Both test tubes are then tapped and placed back into the ice bath for 15 minutes. While waiting, another test tube is obtained, filled with 35µL of competent cells and labeled NP for no plasmid. A water bath is preheated to 37 degrees Celsius and all three labeled test tubes are inserted into the bath for five minutes (Alberte et al., 2012). Using a sterile pipet 300µL of nutrient broth are inserted into both the control and Lux test tubes and 150µL are inserted to the no plasmid test tube to increase bacterial growth. All three test tubes are then incubated at 37 degrees for 45 minutes. Six agar plates are obtained and labeled to correspond each test tube, three of the plates contain ampicillin. A pipet is used to remove 130µl from each test tube containing a plasmid and insert it into the corresponding agar plate. For this, a cell spreader is first

The hypothesis above tested the insertion of the pGlo gene to see if the bacteria, E.Coli, will reproduce and grow in the presence of ampicillin and to see if it will cause a green fluorescent glow. (PGLO™ Bacterial Transformation Kit,2017). Based upon the results from this experiment the hypothesis did support the hypothesis and that the presence of the pGlo gene inserted into the E.Coli did cause for growth and fora fluorescent glow to occur. In the experiment, the petri dishes that contained no pGlo (-pGlo) did not show any reproduction nor did a green glow appeared in both dishes. Unlike the two petri dishes, that contained the pGlo gene and ampicillin, the data data showed both reproduction and a glow in the petri dishes.

In preparing for the bacterial transformation, DNA plasmid is introduced into the E. coli cells that will express newly acquired genes. Two tubes were used and labeled both as +pGLO and -pGLO. A solution of (CaCl2) was transferred 250 µl onto the two tubes. The tubes were placed on the ice. A sterile loop was then used to gather a single colony of bacteria from a starter plate. Now, that both tubes contain bacteria they were placed on the ice for 10 minutes. Four agar plates were labeled as: +pGLO LB/amp, +pGLO LB/amp/ara, +pGLO LB, -PGLO LB/amp. Heat shock was used to transfer both the +pGLO and -pGLO, at exactly 42°C. Time was observed for 50 seconds and quickly return the tubes to the ice for another 2 minutes. As the tubes, cold down they

The two most important sequences on the plasmid that will be used are the ORI bacteria and the antibiotic resistance. The ORI is the origin of the bacteria and is an important part of the bacteria because it does the job of replication. The antibiotic resistance is equally important because it is used to see which bacteria take in the plasmid. In the lab there will be two micro test tubes used. One tube will be labeled +plasmid and the second tube will be labeled -plasmid. Both test tubes will contain the E. coli bacteria and 0.25mL of calcium chloride (CaCl2), but only the +plasmid tube will receive 10pL of pGLO plasmid. There will also be four agar dishes used in this experiment. They will be labeled as follows: LB/Amp+, LB/Amp-, LB+, and LB-. The LB/Amp+ dish will have luria broth, ampicillin, and the pGLO plasmid. The LB/Amp- dish will contain luria broth, ampicillin, and will not contain any plasmid. LB+ also contains luria broth, will not contain ampicillin and will contain the plasmid. The last dish, LB-, will have the luria broth, and will not have neither ampicillin nor the plasmid inserted. The LB/Amp- and LB- dishes act as the control group because they will be used to observe changes that occur without the plasmid. The LB/Amp+ and LB+ will act as the experimental dishes because the plasmid will

After that, a new sterile loop was used to immerse the pGLO plasmid DNA stock tube into the +pGLO tube but no into the – pGLO tube. Both tubes were well closed and put back on the ice for 10 minutes. While both tubes were sitting on ice, the four plates were labeled. LB/amp plate was labeled +pGLO, LB/amp/ara plates were labeled +pGLO, LB/amp plate was labeled – pGLO, and LB plate was labeled – pGLO. After that, the tubes were transferred into a water bath set at 42° C for exactly 50 seconds, then placed rapidly back on the ice for another two

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 LB Plate acts as a control for the LB/AMP because it shows that the bacteria without the plasmid, but still went through the process, survived. The second plate, which contained LB/AMP, left no trace of bacteria behind. This is because the AMP is an antibiotic, which kills off the existing bacteria (LB). This plate also acts a control because without the plasmid, the bacteria can’t grow in the presents of Antibiotics.

coli colony through the process of heat shock to observe differences in growth and bioluminescence with and without GFP and arabinose. It is expected to see no growth in the colonies that have not been treated with ampicillin and pGLO, the plasmid containing the genes for GFP and ampicillin resistance. Those that have been treated with pGLO and/or no ampicillin will grow due to the inheritance of the ampicillin resistance gene, whether or not there is arabinose present. The hypothesis for this experiment is that only the colony that has been treated with pGLO, arabinose, and UV light will be bioluminescent because all are required to glow.

The pGLO plasmid also contains genes that make it resistant to ampicillin which would normally kill E. coli. In addition to this, in order to activate the gene which makes the organism glow there must be arabinose present.

In this lab, Escherichia coli is the experimental bacteria and transformed by pGlo plasmid. The pGlo plasmid from Bio-Rad contains the coding genes for Green Fluorescent Protein (GFP) and a gene resistant to the antibiotic, ampicillin (Mardigan, 2011a). Normally, the GFP is coded from a gene in the jellyfish, Aequorea Victoria. The GFP can also only produce its green fluorescent color when the chromophore is in a specific conformation. When the GFP is denatured, the chromophore loses its original shape thus losing its fluorescent capabilities.

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

Hypothesis: If genetic transformation involves the insertion of a gene into an organism in order to change the organism’s trait where genes can be moved from one organism to another with the aid of a plasmid and pGLO plasmid encodes for a gene resistant to the antibiotic ampicillin as well as the gene GFP which allows the bacteria, E. coli, to express the glowing gene by producing the fluorescent protein if switched on after having undergone transformation by adding sugar arabinose – which is a nutrient for the cell -, then when the procedure of the pGLO transformation lab is completed as instructed in the student manual, the agar plate +pGLO Lb/ampicillin will produce colonies of bacteria that do not glow, -pGLO LB/ampicillin will not produce any bacteria, and –pGLO LB (constant) will produce a lawn of bacteria that does not glow (instead appearing as a

If Genetic transformation has the meaning of “change caused by genes” and involves the placing of a gene into an life form in order to modify the organisms characteristic; the progression of placing genes from one life form to a different is used to assist of a plasmid and the pGLO plasmid codes the gene used for GFP as well as the gene for resistance to ampicillin. It is used to manage the expression of the fluorescent protein; hence, the GFP gene is able to be switched on by adding the sugar arabinose to nutrient medium of the cell, then the bacteria will be able to glow a bright green underneath UV light when arabinose is within the nutrient agar medium. Hence, then when one micro test tube +pGLO and –pGLO are labeled and placed into a foam rack and the tubes are open and using a sterile pipet used to transfer 250 micro liters of transformation solution (CaCl2 ) in each tube, position the two tubes on ice, pick up 2-4 colonies of bacteria with a loop, submerge the loop into the +pGLO tube, repeat steps for –pGLO, put in to ice, and put plasmid DNA into the pGLO; after the pGLO’s need a heat shock by placing the cold tubes into the 42 degrees Celsius hot bath for 50 seconds and back into ice for 2 minutes, later insert the 250 micro liters of LB nutrients broth into the tube and then placing 100 micro liters into the 4 plates, each individual plate contains +pGLO LB/amp, +pGLO LB/amp/ara, -pGLO LB/amp or –pGLO LB). If bacteria that contains +pGLO plasmids is resistant to