Transformation of Escherichia coli in different concentrations of Plasmid DNA
This report discusses an experiment which students have to transform and plate competent Escherichia coli in different concentrations of plasmid DNA. This experiment uses four concentrations of plasmid DNA to perform four transformations. These concentrations are namely, 5 µl of distilled water, which acts as the control in this experiment; 2.5 µl of undiluted plasmid DNA; 1.0µl of undiluted plasmid DNA; and lastly, 1.0µl of plasmid DNA diluted 1 in 10. Transformation is an important aspect in genetic engineering as it allows for a particular DNA to enter into another cell. The aim of this experiment is to be able to perform a cell transformation …show more content…
Then, we placed 100 µl of the E. coli cells into four Eppendorf tubes each and labelled them A, B, C and D. After filling them up, we placed them into an ice box. After this, we needed to perform the transformation process by mixing the E. coli cells with different concentrations of plasmid DNA. In test tube A, we added 5 µl of distilled water to the E. coli cells. In test tube B, we added 2.5 µl of undiluted plasmid DNA to the E. coli cells. In test tube C, we added 1.0 µl of undiluted plasmid DNA to the E. coli cells. Lastly, in test tube D, we added 1.0 µl of plasmid DNA diluted 1 in 10 to the E. coli cells. Then, we used a P100 pipette and mixed all the liquids in all the Eppendorf tubes by pipetting the solution up and down. Then, we left the Eppendorf tubes along with its contents in the ice box for twenty minutes. After twenty minutes, we labelled the base of the four agar plates to match with the appropriate plasmid DNA concentration about to be plated. We also wrote our initials, the date, and the table number on the base of the agar plate. We then gently lifted up the cover of the agar plate with just enough space to allow us to plate each tube of cells onto four different agar plates – one agar plate to one plasmid DNA concentration. We used a spreader to spread the cells evenly over the surface of the agar, being careful not let the spreader touch
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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.
In order to find transformation efficiency, the total number of colonies on the plate is divided by the total amount (µg) of DNA spread on the plate. All the factors that must be taken into account while finding the transformation efficiency include; the total amount (µg) of plasmid DNA used, the total volume (µl) of cell suspension prepared, the fraction of DNA spread on the plate and finally the total amount (µg) of DNA present on the plate. In the LB/AMPC plate the transformation efficiency was 8.2 x 103 colonies per µg of plasmid DNA while the LB/AMPlux plate the transformation efficiency was 1.07 x 104 colonies per µg of plasmid DNA. The transformation efficiency for the LBC and LBlux plates were not taken into account, even though they too transformed because they were not in a restrictive environment where they needed to express their ampicillin resistance genes in order to
This lab is about moving genes from one thing to another using plasmids. Plasmid has the ability to replicate, so it replicates independently, and separately from the chromosomal DNA. Plasmid are one or more small piece of DNA and they enter cells as a double strand DNA. When they enter the cell as a doubke strand they do not invade he chromosomal DNA. We will also transform bacteria into GFP which is mainly from the jelly fish Aequorea Victoria. The GFP causes the the jelly fish to fluorescent and glow in the dark. After the transformation, bacteria starts to make the GFP which causes them to glow a green color under a ultraviolet light.
E. coli HB101 was transformed with pGLO plasmid then grown on media containing ampicillin and/or arabinose and on medium containing neither (Brown, 2011). This is done for selection of transformed cells since not all cells are expected to take up the plasmid (Brown, 2011). We also expect roughly the same CFU on any plate(s) receiving samples from the same microcentrifuge tube, since they are getting the exact same
The instructor split the class into two separate groups one with the plasmid lux and the other with the plasmid pUC18. Group two was assigned to test the lux plasmid. The, Eppendorf, tubes were labeled “C” for the control plasmid DNA and “lux” for the plasmid lux DNA. The two tubes were then placed into the ice bath. Using a sterile micropipette 5 uL of the lux plasmid was added to the tubes labeled “lux” or 5 uL of the control plasmid was added to the tubes labeled “C” for the control plasmid DNA. Eppendorf tubes had 70 uL of the competent cells added to them with a different transfer pipet. All the tubes were then stored in the ice bath for about fifteen minutes. Another test tube was labeled “NP”, which stands for “No Plasmid”, and 35 uL of competent cells was added to each of the test tubes labeled “NP” during the fifteen minutes. Once the fifteen minutes are up, all three tubes were placed into a preheated water bath at 37 °C for about five minutes. To both the lux
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.
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 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
Spin the two tubes in a centrifuge for 5 minutes on opposite side of the centrifuge. The bacterium will collect at the bottom of the tube, so pour out the extraneous supinate. Then, add 250 microliters of buffer. The Ca2+ cation of the buffer neutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane allowing the DNA to pass through the cell wall and enter the cells. Place both tubes on ice. Then add 10 microliters of water into one tube and 10 microliters of plasmid DNA into another tube labeling the one with DNA with a + and the one with water -, and place on ice for 10 minutes.
One of the most imperative functions in maintaining the development of evolution is the frequency of genetic transformation: the injection of foreign DNA into another organism’s DNA. This term is defined by the actions of a vector, but more specifically by the actions of plasmids and phages. However, in this experiment we are primarily focused on the effect of the pGLO plasmid transformation of GFP on the E. coli bacteria by introducing a second chromosome or a plethora of cloned plasmids. (Bassiri 2011)
For this experiment, E. coli was best for genetic engineering because of their size, and their fast reproduction (Spilios, 2017). E. coli will be genetically transformed using an engineered plasmid. A plasmid is a circular piece of DNA which independently replicates and multiplies because it has its own origin of replication (Spilios, 2017). The pGLO is the plasmid used in this experiment. Plasmids are used as vectors and they contain manipulated genes such as genes coding for antibiotic resistance for drugs like ampicillin. This antibiotic resistance of such serves as the selectable marker in genetic transformation and for genetic transformation to proceed, the cell must reach competency which is the physiological state that is required for the vector plasmid to get into the cell for transformation (Spilios, 2017). While competency can be reached naturally in some organism, it must be reached artificially in E. coli through treatment with CaCl2 and exposing them to heat shock using incubation (Spilios, 2017).
If a gene that codes for Green Fluorescent Protein transforms bacteria and GFP glows when transformation occurs, then when two micro test tubes have 250 microliters of transformation solution and places in an ice bath, then 2-4 bacteria colonies are added to each tube with a sterile loop; then a plasmid (pGLO) is added to one of the tubes, incubated in ice for 10 minutes, then heat shocked for 50 seconds at 42 degrees Celsius, then back into 9ice for two minutes; then LB nutrient broth is added to both tubes (250 microliters) and set out at room temperature for 10 minutes. Then, 100 microliters of each solution in the tube are added to four
Bacterial transformation is the process of moving genes from a living thing to another with the help of a plasmid.The plasmid is able to help replicate the chromosomes by themselves; laboratories use these to aid in gene multiplication. Bacterial transformation is relevant in everyday lives due to the fact that almost all plasmids carry a bacterial origin of replication and an antibiotic resistance gene(“Addgene: Protocol - How to Do a Bacterial