E. Coli Cell Genetic Transformation

By subjecting these bacteria through sudden temperature changes or heat shock, a difference in pressure in the outside and the inside of the cell is made, that causes pores, through which the mobile supercoiled plasmid enters. After normalizing the cell temperature, its walls will heal. When the E. coli has now taken up the plasmid with GFP, it will be able to grow in agar plates with ampicillin, an

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

coli, to express a gene that is normally causes jellyfish to be fluorescent. The plasmid that contains the gene for the green fluorescent protein (GFP) is called pGLO. The type of genetic transformation we used in the experiment was heat shock which made the membrane of E. coli fluid and allowed the pGLO to enter the cell. In order for the E. coli to express the GFP gene there needs to be arabinose presence, which is a sugar that acts as an on switch for the pGLO. Using this information, we predicted the bacteria E. coli will grow and express the GFP on the plate that consists of +pGLO, ampicillin, arabinose, and LB broth (Luria Bertani broth).

Griffin was a scientist that showed that there was a non-living component to heritability between organisms, although he had not yet discovered what exactly was the heritable component. However, during this time period, it was a common belief that the heritable material was contained within the protein of the organism, not the DNA that is now held in the truth. In the Avery and company experiment, they expanded Griffin research to pinpoint the exact location of the heritable information by breaking down cell components until the final product allowing transformation was

You want to make sure they are sterilized so no other bacteria can get in the way of the experiment. It is also important to was your counter space with bleach and wash your hands very well. Once that is all done, take both of your micro test tubes and name one –pGLO and the other +pGLO, then place them in the foam rack. Use a sterile pipet to transfer 250 microliters of CaCl2. Place the tubes on ice for however long you think it will take for them to get cold. After however long you wish to have the tubes on ice, use a sterile loop to pick up a single colony of bacteria from the plate that will be passed around the classroom. Start with the +pGLO tube and put the loop in the plate, spin the loop between your index finger and thumb until you get enough of the solution and that it is all off the loop. Repeat this step with the –pGLO tube, but with a separate sterile loop. Put the tubes back on the foam rack and immerse the rack in ice for ten minutes.

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.

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

While conducting this experiment many questions came to mind what effect can plasmids had on the transformation of E. coli and why did CaCl2 had to be used. During the experiment the solution was used to neutralized negative charges and heat sock was done to traumatize the cell membrane to get the cell ready for the intake of plasmids. As shown above observation can be made from the agar plate which describes the affect that plasmid had on the each. First, with the LB c, LB np and LB lux DNA exhibited lawn growth consequently lux brought about the bioluminescence effect on plates LB lux. This was the result of the absence of the Ampicillin antibiotic and the lux together. So, agar plates labeled LB/AMP c and LB/AMP lux, shown colonial growth which is a clear indication that minimal

Genetic engineering and transformation is the “modification of an organism's original genetic composition by artificial means”(Genetic Engineering 2016). Genetic engineering and transformation involves the swapping of traits and genes between organisms to create a genetically modified organism or GMO. Genetic engineering has it’s advantages which include the prevention of disease by isolation of the gene that causes the disease, being able to take the “desirable traits” of one organism and implanting them into another, and genetic engineering can increase diversity in plants and animals by creating alleles that can be given to different species of organisms according to the Asia-Pacific Economics Blog (Apecsec.org 2014). Unfortunately, genetic

The bacteria that was used in the experiment was Escherichia coli which also has the ability to communicate through quorum sensing. Quorum sensing controls bioluminescent, antibiotic production, competence this better understand the bacteria that we were working with (Bassler, 2012). We wanted the bacteria to express antibiotic resistance, bioluminescent and growth all of that is control by quorum sensing which is a type of communication between the populations of the Escherichia coli. In addition, Escherichia coli is a gram negative bacteria which appear as purple when is stained. Gram negative have a thin layer of peptidoglycan but the outer membrane has porins which lets antibiotics such as ampicillin to move through the wall and the bacteria can become resistant to antibiotics. In the experiment, one of the way we made the Escherilia coli was make them competent to allow them the express and share the important information between themselves in the form of genes in the selected plasmid. Plasmids help the bacteria to transfer important date for its survival and its one of the features of the plasmids that is used in the transformation. The Escherilia coli first needed to become competent to take the DNA with the help of calcium chloride

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.

In this experiment the goal was to insert the plasmid pBI121 to our E. coli cells. This was accomplished by making our E. coli cells with plasmid competent via heat shock. Our cells were then inoculated onto a kanamycin plate and incubated at 37 degrees Celsius for a day. Our next lab period we preformed a mini prep and extracted our plasmid, through a series of buffers and centrifugation. Once we obtained our isolated plasmid we then preformed a restriction digest and added two restriction enzymes to our plasmid BAM and SAC and preformed a gel electrophoresis. In theory we should have seen two bands, one at the 2kb and the other at 14.7kb. If

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.

This experiment was performed to test the hypothesis if LB nutrient broth, +pGLO and -pGLO Ampicillin, and Arabinose was placed in the E. coli plates, then there will be a significant growth in the newly transformed bacteria and it will possess the ability to glow under UV light. The measurements were recorded from the bent glass tube in each glass test tube. The transformation protocol tested for the newly possessed traits in E.coli bacteria. Throughout the experiment there were many probable reasons for failure. If the pipettes and sterile loop were not thrown out in between each use, a cross contamination could cause a miscalculation in the experiment causing the data results to fail. The hypothesis that was tested was validated due to the positive results with each experiment stating that newly transformed organisms due in fact pass on traits.