Dean 1 Sara Dean Mr.

Wan AP Biology 5th 18 JAN 2013

Transformation Lab Questions 1. Under the UV light, only the colonies on the + plasmid plate with ampicillin glowed. The total colonies for all plates were: + plasmid w/ ampicillin 117 colonies + plasmid w/ no ampicillin 103 colonies - plasmid w/ ampicillin 87 colonies - plasmid w/ no ampicillin 122 In the lab, only the + plasmid tube is supposed to receive the plasmid solution, but our group had suspension added to both the + and - plates by the instructor as a test. 2. Yes, I hypothesized that all plates would glow and show colonies, due to the extra plasmid solution being added to both tubes, resulting in colonies on the - plasmid plates. 3. A. Since there are no colonies at all, even on plates without the resistant (antibiotics), the plates either began with no bacteria, or the bacteria died and were unable to survive in the conditions it was placed in. Also, the bacteria could have been killed by the heat shock. B. On the ampicillin plates, the lawns show that the gene was taken up and the bacteria was able to survive. The clean kan plates are clean, meaning the kan gene was never accepted, or possible even added, to the bacteria/plates. Lastly, the LB broth plates show a lawn as well, meaning the bacteria was able to survive on these plates. C. The colonies (rather than lawn) on the ampicillin plates show that the some, but not all, of the bacteria accepted the gene. The clean plate result of the kan experiment shows that the gene was never accepted or possible even added to the bacteria. For the LB plates, the lawn shows that the bacteria were able to survive and thrive in this environment. 4. A. 10 uL of plasmid x 0.005 ug/ul = 0.05 ug number of colonies/0.05 ug = transformation efficiency

B. 117/0.05 2340

Dean 2

C. By using an inexact transfer method (micropipette), the exact amount of plasmid may be inaccurate. Also, adding and mixing the plasmid (possibly inexact) might have slightly changed the ratio as well. 5. 2/402 mL x 10 mg/mL = 20/402 mg/mL x 10 ug/mg = 200/402 mL

6.

1. 2.

3.

4. 5.

Big Idea Assessments In this experiment, the gene pGlo directly changed the phenotype of the gene in the experiment by making it begin to glow under a UV light. The protein of the gene is now expressed, with its DNA, directly by the bacteria. The bacteria that was already alive had to transform to accept the pathogenic gene, which is what ultimately made the mice sick. The phenotype was changed (from rough to smooth in this particular experiment) due to the gene controlling the protein that affects whether the bacteria were smooth or rough. DNA was decided the molecule that passed on traits when it became evident, especially in Griffiths experiments, that bacteria could only transform when DNA was present. The experiments used the fundamental ideas of depriving bacteria of protein and RNA, and seeing no transformation; while when DNA was present, the bacteria did transform. To introduce and accept the gene, the bacteria would need to be heat shocked and then cooled. Following a similar laboratory to this one, the gene could be accepted. The two ways a bacteria can acquire new genetic material is through transformation, and transduction. For transformation, DNA must be present and the bacteria must not only accept the introduced gene, but must be in an ideal environment to thrive and transform. For transduction, a virus must be injected with the DNA.

6.