Conjugation and Transposition in Escherichia Coli

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 determine which ones would be resistant, intermediate, and susceptible to our unknown strain of E. coli we had three antibiotics and one control per agar plate, and we replicated this twice. A lawn of the E. coli strand was spread evenly on each agar plate with a cotton swab. We then divided the agar plate into four halves and placed an antibiotic sample in each half with forceps. We incubated the dishes for forty-eight hours. Our independent variables are the different types of antibiotics with our known and unknown E. coli that we used to conduct the experiment and our dependent variables are the diameters of the zone of inhibition for each antibiotic.

As predicted the E. coli colony transformed with either the PUC18 or the lux plasmid developed an ampicillin resistance. Which made it easier for them to not only survive but also replicate in both the LB agar plates and the LB ampicillin rich agar plate. However the E. coli colony not treated with the plasmids could not survive and colonize in the LB ampicillin rich agar plates. The plate that had no ampicillin in its environment and no plasmid treated E. coli served as a positive control for this experiment because it demonstrated how the E. coli would colonize and grow in a normal setting. The cells in the positive control plate grew into lawn colonies because they were not placed into a selective environment or transformed, so they had no need to acquire ampicillin resistance. Two plates in the experiment contained E. coli cells that were transformed with either the PUC18 or the lux plasmid but were placed in an ampicillin free environment. These two colonies grew

First, 50 uL of bacterial cells were made competent by being treated with 590 uL of CaCl2 in three different test tubes before transformation of the plasmids took place. In order to enhance the uptake of the plasmid DNA the instructor added the CaCl2 solution to the bacterial cells. The tubes were placed into a cold ice bath for about ten minutes after the CaCl2 solution was added into the test tubes of E. Coli.

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 was to show the genetic transformation of E. coli bacteria with a plasmid that codes for Green Fluorescent Protein (GFP) and contains a gene regulatory system that confers ampicillin resistance. A plasmid is a genetic structure in a cell that can replicate independently of chromosomes. In this lab, the Green Fluorescent Protein, which is typically found in the bioluminescent jellyfish Aequorea Victoria, was cloned, purified, and moved from one organism to another with the use of pGlo plasmids. It was hypothesized that if bacteria that were transformed with +pGlo plasmids are given the gene for GFP, then transformed cell colonies

Escherichia coli, or E. coli, is a common bacterium that can be found in diverse environments all over the planet, including the gastrointestinal tracts of animals and humans. Many of these strains of E. Coli are essential mechanisms in the digestive tract, while others are pathogens that can cause complications in urinary and intestinal tracts. (Payne & Sparks) In research, E. Coli is commonly used as a model organism, meaning they are widely studied by scientists for a variety of purposes due to their experimental advantages. E. Coli is comparatively simple, and there are many advantages to studying these prokaryotic cells in the fields of biochemistry and molecular biology. E. Coli has this simplicity and is relatively easy to propagate in a lab environment. Their genome has been completely sequenced and many things we know about DNA, protein synthesis, and gene linkage have been derived from studies regarding this particular organism. (Cooper)

Smith H. 1969. TRANSFER OF ANTIBIOTIC RESISTANCE FROM ANIMAL AND HUMAN STRAINS OF ESCHERICHIA COLI TO RESIDENT E. COLI IN THE ALIMENTARY TRACT OF MAN. Science Direct [Internet]. [Cited 2015 Dec 1]. Available from:

Figure xxx: The total plate count of the Escherichia coli from day one to seven.

The purpose of this experiment is to show how different concentrations of Ampicillin affect Escherichia Coli Growth and how the bacteria become resistant to the antibiotic drug. Through a series of steps, which involves streaking agar plates with E.coli sample and application of ampicillin to the E.coli sample on the agar plate, the experiment yields a result that supports the hypothesis. The hypothesis acclaims that ampicillin would affect the growth of E.coli; measuring the zone of inhibition approves the claim in the experiment. The measurements of the zone of inhibition indicate that the generation of E.coli expands as the radius reduces. The reduction of the radius shows the E.coli population is reducing and becoming resistant. In other

Abstract:Conjugation is a natural occurring process that involves the transfer of DNA from one cell into another through a physical connection between the cells. In the following experiment, two strains of Escherichia coli bacterial cells (donor F'lac+strs and recipient F-lac-strr) underwent conjugation to produce a transconjugant strain (F'lac+strr). MAC plates and streptomycin were utilized to determine if conjugation had occurred. When plated, the donor colonies appeared red and the recipient colonies appeared white. The transconjugant plates showed red and white colonies. Using alkaline lysis miniprep, a DNA plasmid was isolated from the donor and transconjugant strains and FIGE electrophoresis was used to determine the size of the

This experiment was carried out by mating Hfr lac- mutants (CSH61) to three F- strains (CSH54, CSH55, and CSH56) with deletions in the lac-pro region, each with a different suppressor and a type of antibiotic resistance. Mating would allow for transfer of the lac-pro region from the Hfr

Elements belonging to KPC transposon Tn4401, including tnpA, tnpR, ISKpn6, and ISKpn7 (14), were identified by PCR and sequencing on both KPC-3–encoding plasmids originating from Kpn1 and Eco2. These genetic determinants were absent in the susceptible Eco1. These data suggest that Eco1 has acquired pKpQIL from Kpn1 in the patient’s gut, leading to the formation of Eco2. Although acquisition of the plasmid increased MICs for imipenem, meropenem, and ertapenem considerably, it did not confer full resistance (Appendix Table) presumably due to copy number of the plasmid or the expression level of blaKPC-3 in E. coli. Curing of pKpQIL from Eco2 was performed by sequential transfers at an elevated temperature (42°C). The cured strain, which lacked the KPC-encoding plasmid, showed full susceptibility to all antimicrobial drugs tested, similar to the Eco1 strain isolated from the patient’s gut flora.

The genomic DNA sample concentration of 28.5 ng/μL and its A260/A280 ratio of 1.85 indicates that the DNA was relatively pure since a 260/280 ratio of ~1.8 is generally accepted as pure for DNA (Cox, Doudna & O’Donnell, 2015). The ratio of white colonies to blue colonies observed on the blue-white screen plate was 31:37, which shows that there was a greater number of E. coli DH5-α that did not contain the plasmid with the foreign gene insert. Therefore, out of all of the E. coli DH5-α cells that were plated and were transformed successfully, there were more cells containing the plasmids without the foreign insert than there were plasmids with the foreign insert.

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