Biotechnology Lab Report On E.Coli
Biotechnology has been used to produce more food like wine and cheese, but recent years of biotechnology has been so advanced that provides products like vaccines detergents and drugs. Bacterial transformation is a form a biotechnology. For this lab we used a common bacteria in our digestive tract named Escherichia coli or E. coli for short. This is used in labs because of its instantaneous growth. Ampicillin is a destroying antibiotic for E. coli, and if the cells contain the resistance gene the cells can survive with the existence of ampicillin since the ampicillin that inactivates the antibiotic. The purpose of this lab experiment is being able to understand the effects of bacterial transformation with different environments and to see the blue color change from the pBlu plasmid. If the transformed E. coli is mixed with the ampicillin resistance gene, it will be able to grow in the ampicillin plates and the non-transformed E. coli will not. Our group hypothesized that the E. coli the plates with and without plasmid that receive the broth would reveal growth. We also believed that the positive control plates will grow because of the resistance gene and he negative control will die off. The ampicillin and X-Gal will die in the negative control with no plasmid that obtained broth due to that it's not resistant. The ampicillin and X-Gal will turn blue in the positive control with the plasmid that was obtained.
Methods
Student were provided gloves for safety. Before the lab began we made sure we are working with a clean environment, where no cross contamination occurs, which makes organization is a factor. Using a sterile pipette, we added Calcium chloride solution to each tube and both was immediately placed on ice. We transferred bacteria from the plate to the "positive plasmid" using a sterile inoculating plastic loop. Submerge the loop tip into the calcium chloride solution in the "positive plasmid" tube and stir the loop fast to remove the cell mass and spread the entire mass into the calcium chloride solution. For best results we made sure no clumps of the cells were present. Once we completed that step the tubes were set back on ice.
We repeated certain steps and use a new sterile loop to transfer a mass of cells to the "negative plasmid" tube. Both tubes will be placed on ice now. In the "positive plasmid" we used a sterile inoculating loop to pick up one loopful of plasmid DNA and added into the calcium chloride, then mix. Next we put back the "positive" tube to the ice. There should not be any traced of plasmid in the "negative" tube. Incubate both tubes on ice for roughly 15 minutes. After the incubation, "heat shock" the cells, then carry the ice cup with the solution to the 42°C water and place in the water for 90 seconds. After heat shock, then return the tubes to ice and let them remain on ice for at least one minute. The tubes was properly stored for next class and removed just prior to the beginning of the next lab. Labels were added to the bottom of your broth/Amp plates. Using a pipette we added cell suspension from the "negative plasmid" tube. For each plate spread the suspensions evenly on the surface of the agar by spreading the surface of a new sterile across the plate surface. Using a pipette to relocate of cell suspension from the "positive plasmid" tube to the appropriate plate and spread as above. Rest the plates on the bench for 10 minutes to allow the cell suspension to absorb. Tape or wrap around your four plates to seal the lids from contamination. Place the plates upside down in an incubator or at room temperature. The results were ready to observed in 24 hours if incubated at 37oC. Due to class days results were shown the following week.
Results
Our results provided that the negative control with the ampicillin and X-Gal had died.
