This lab deals with the process of genetic exchange in prokaryotes. There are three main mechanisms of genetic exchange which include transformation, transduction, and conjugation. In transformation, DNA is released from cells in the surrounding environment which is then incorporated into the recipient cells DNA. In transduction, DNA is transferred through a virus to the recipient. In conjugation, genetic exchange occurs through direct contact with another cell and the plasmid is transferred from the donor to recipient. Plasmids are circular modules of double-stranded DNA that are beneficial but not essential. R factors are plasmids that carry genes that confer resistance to antibiotics on the host cell. R factors have been a problem because they are causing many strains of pathogenic bacteria to be highly resistant to antibiotics.
The transformation was the first mechanism of bacterial exchange that was discovered. A famous experiment with transformation dealt with injecting mice with an avirulent strain of bacteria with heat-killed cells of a virulent strain killed the mice while injecting these strains separately did not. This established that the surviving cells were recombinant. A genetic exchange of the DNA in the external medium had occurred between the dead cells and the live ones. The bacteria that we are using is E. coli bacteria which are capable of being artificially transformed. They are made competent (capable of being transformed) only after following the subjection of cells to calcium chloride solution.
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Transformation of E. Coli
A. Summary – In this lab, we are investigating the method of genetic exchange called transformation through the insertion of plasmid pUCB DNA, which carries the gene for antibiotic resistance to ampicillin, into competent E. coli cells.
B. Procedure – The procedure of this lab is somewhat complicated. 250uL of calcium chloride to 2 separate tubes labelled + and –. Next, transfer a large colony of bacteria from the starter plate to the tube of cold calcium chloride and twirl rapidly. Add 10uL of the plasmid solution to the “+” tube. Then, incubate both tubes on ice for 15 minutes. During this time, obtain 2 Luria agar plates and two Luria agar plates with ampicillin. Label one plate “+” and the other “–“. Next, remove the tubes from ice and immediately place them in a hot water bath for 60-90 seconds. Remove the tubes and place them on ice for 2 minutes. Mix the Luria broth with each tube and add each solution to the labelled plates. Spread the cells over the entire surface of the plates with the bacteria spreader. Label and let the plates sit for 5 minutes then place the places inverted in an incubator overnight.
C. Findings – After staying up all night thinking about the incubation, we finally got to record the number of colonies if there were any. It turns out that we didn’t have any colonies but the bacteria had grown over the entire surface of the plate which is referred to as a “lawn”. These were our findings: the plate without ampicillin and with the plasmid grew a lawn, the plate without ampicillin and without the plasmid grew a lawn, the plate with ampicillin and with the plasmid also grew a lawn, and the plate with ampicillin and without the plasmid had no growth. We also calculated transformation efficiency and the following table were our calculations:
The total mass of plasmid used
The total volume of suspension
A fraction of cell suspension put on a plate
The total mass of plasmid infraction
Number of colonies per ug of plasmid
D. Conclusion – So basically, a transformation occurred and we know this because the plate with the ampicillin and the plasmid showed growth whereas it wouldn’t show growth if transformation did not occur. There are many possible sources of error which include human error (not following directions) and there could be an error if the ampicillin was actually penicillin and the company that makes the labs is involved in a large scandal to rip off schools. It could happen and about a million other things could go wrong.
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