Pglo Report

In the LB (pGLO negative), it is expected to not see any colonies growing. As a result of this experiment, it shown any growth colonies but this only had shown a large number of white, circular colonies that were found across the surface of the agar. In the LB/Amp (pGLO negative), it is expected to see any growth colonies. In the experiment, the resulted was no growth colonies because this has Ampicillin and no pGLO. Now, the LB/Amp (pGLO positive), it is expected to have growth colonies in the agar plate. As a result, it was shown growth colonies in the agar plate because has positive pGLO regardless having Ampicillin. In the other hand, both positive pGLO have the same components but in one plate was added Arabinose. The LB/Amp/Ara (pGLO

This experiment was designed to test and observe the transformation efficacy of the pUC18 and lux plasmids in making E. coli resistant to ampicillin. Both plasmids code for ampicillin resistance, however, the lux plasmid codes for a bioluminescence gene that is expressed if properly introduced into the bacteria’s genome. The E. coli cultures were mixed with a calcium chloride solution and then heat shocked, allowing the plasmids to enter the bacteria and assimilate into the bacterial DNA. The plasmids and the bacteria were then mixed in different test tubes and then evenly spread onto petri dishes using a bacterial spreader, heating the spreader between each sample to make sure there is no cross contamination. Each of the dishes was labeled and then incubated for a period of 24 hours. The results were rather odd because every single one of the samples grew. Several errors could have occurred here, cross contamination or possibly an error in preparation as every single sample in the class grew, meaning all samples of the bacteria transformed and became ampicillin resistant.

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

An organism's traits are often caused by a combination of its genes and the environment it lives in. Think about the green color you saw in the genetically transformed bacteria:

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

First, 500μl of calcium chloride was added to a DNA tube. Calcium chloride makes it easier for cells to transform. Approximately 15 E. coli colonies were then transferred to the tube. 250μl of the DNA was transferred into another tube, where 10μl of pGFP was then added. The plasmid for green fluorescent protein is what will be transformed into the E. coli’s DNA so it will express the GFP. The DNA tubes were then incubated in hot and cold water .This process is called “heat shocking” and affects the permeability of cells, allowing for new DNA to move into cells more easily. Initially the tubes were put in ice for 10 minutes, they were then put in water that was 42℃ for 90 seconds, after that they were put on ice for another 2 minutes, 250μl

During this lab, genetic transformation was looked at. Genetic transformation is the insert of foreign DNA into a host cell. Competent cells are cells that can take up genetic transformation through the vector, which is the transfer mechanism of a gene. In lab, the competent cell that was used was E. coli and the vector is pGLO. The method heat shock was used, which is the sudden changing of temperature that increases the absorption of the plasma membrane, making the cells then have to pick up the DNA from other medium. E coli was the first gene to be heat shocked and is known as a good heat shock protein, it was first uncovered in 1991. (Lai et al. 2014) Here, the bacteria E. coli is being transformed by the gene encoding green fluorescent protein (GFP).

Plates one and three represented our control plates for this experiment. Plate one was Amp negative and plasmid negative, therefore it was a normal E. coli strain growing on a plain nutrient broth agar plate. This plate provided us with a baseline that showed what normal E. coli looks like growing on a normal plate after being exposed to a stressful environment. Plate three was also considered another control plate because it showed how viable a plasmid negative E. coli strain could be on an Amp positive plate. We can use this plate to compare how well a plasmid positive E. coli strain will survive on an ampicillin positive plate.

Mark one tube “+” and the other “-”. The “-” tube will include the plasmid. Using a sterile graduated pipet, add 0.25 mL ice cold calcium chloride to each tube. Hold those tubes on ice. Obtain a starter plate of E. coli and using a sterile inoculating loop to transfer one large colony of bacteria form the starter plate to each tube of cold calcium chloride. In order to remove the bacteria from the transfer loop and break them apart, place the loop into the calcium chloride and twirl the loop rapidly. Dispose of the loop in a bio hazardous waste. Then, using a sterile inoculating loop, dip the loop into the DNA stock tube. When you remove the loop from the solution, check to be sure that there is a drop of liquid contained in the loop area. Transfer the liquid (approximately 10 μL to the “+” microfuge tube and twirl the loop to mix the plasmid into the solution. Place both the positive and negative tubes on ice for 15 minutes. While the tubes are on ice, obtain two Luria agar plates and two Luria agar plates with ampicillin. Label one Luria agar plate “+” and the other “-”; do the same for the Luria agar plates with ampicillin. After 15 minutes, remove the tubes from ice and immediately place them in a 42 degrees Celsius water bath for one minute. Remove the tubes from the water bath and immediately place them back on ice for two minutes. Remove them from the ice bath and add 0.25 mL of room temperature tryptic soy broth to each tube with a

The micropipette is used to collect very small amounts of liquids that an average pipette would not be able to accurately collect. For this lab, one needs to gather two tubes which will each contain 250µL of solution. One tube will then be filled with the +pGLO plasmid and the other will contain -pGLO. The pGLO plasmid is NOT transmitted into the -pGLO tube. The solution the pGLO plasmid is in is called transformation solution and is used to soften the membrane of the E. coli cells with the calcium chloride it contains. The tubes must remain in an ice bath for ten minutes to bring their temperature down so that they will react strongly when exposed to the heat shock. Whenever researchers are transferring the E. coli bacteria from tube to tube, they must wear protective gloves and sterile loops provided to them. Researchers need to obtain and label four plates: +pGLO LB/amp, +pGLO LB/amp/ara, -pGLO LB/amp, and -pGLO LB. Amp stands for ampicillin and ara stands for arabinose. Their plates contain a base gelatin of Luria-Bertani broth. Then, once the tubes are very cold on ice, researchers place the tubes in 42-degree celsius water for fifty seconds exactly in order to activate the heat shock of the E. coli cells, prompting their membranes to become more permeable and susceptible to accepting the pGLO plasmid. After the

For this lab we used two microcentrifuge tubes, a pair of vinyl gloves to prevent contamination, a micropipetter, six sterile micropipetter tips, 500 µl of a transformation solution containing CaCl2, seven sterile loops, ice, a timer, a 42°C water bath, a floating rack two colonies of E. Coli bacteria, pGLO plasmid DNA, four Luria Bertani (LB) broth nutrient agar plates (one with LB alone, two with LB and ampicillin, and one with LB, ampicillin, and arabinose), 500 µl of LB nutrient broth, tape, a UV light, and a 37°C incubator.

pGLO is a plasmid that contains several genes, araC, gfp,bla, and an ori of replication. E. coli was artificially induced that became a competent bacteria when it took the pGLO DNA, so it had the ability to have ampicillin resistance and fluoresced when arabinose was present. Two tubes with E. coli were labeled to differentiate which tube the pGLO was added to, then through several steps the bacteria was induced to intake the pGLO DNA. At the end, each tube was inoculated on to three different plates that contained different substances and they were incubated then observed. The results showed only one E. coli culture had growth and fluoresced which was the pGLO+ E. coli that was grown on the plate with LB, amp +arab, there were only two E. coli cultures that did not grow because the pGLO- E. coli did not have the ampicillin resistance to grow in ampicillin conditions, and the rest of the culture plates showed growth. pGLO could be used in food safety experiments done by the food safety department that helped to identify Salmonella and Yersinia enterocolitica in pig slurry, so they were able to calculate how long these bacteria lived in certain conditions before disinfectants were used.

In the lab conducted, the main idea stemmed from the topics of genetic engineering and genetic transformation. By definition, genetic engineering is the “modification of an organism’s genetic composition by artificial means” (Lerner). This often involves the micro-insertion of genetic material. Genetic transformation is the “genetic alteration of cells resulting from the direct intake of genetic material through the cell membrane” (Transformation (genetics)). Both differ from other means of genetically altering organisms due to engineering being able to occur between highly differing organisms not of the same species, and transformation occurring at a molecular level

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

The materials required for this experiment were 2 micro test tubes, a foam rack, sterile pipette(s), 250mL transformation solution(〖CaCl〗_2), an ice bucket, and 5 plates (1 LB, 2 LB/Ampicillin, 1 LB/Arabinose, and 1 LB/Ampicillin/Arabinose). All of the materials that are listed were used to test the hypothesis. To begin the experiment, the 2 micro test tubes needed to be gathered and labeled with one written as “+pGLO” and the other as “–pGLO”. After that was finished, the labeled test tubes