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Title: Colony Transformation Lab Purpose: To study the behavior of Escherichia coli once it has been introduced to a foreign gene. Hypothesis: If the ampicillin resistant DNA is introduced into the E. coli bacteria, through the uptake of this DNA the bacteria will receive the resistant gene that will permit the bacteria to grow freely in the presence of ampicillin. Experimental Design: By observing the E.
coli bacteria one can study how it reacts when a foreign gene such as the ampicillin resistant gene is introduced. Through the course of this experiment, simply introducing a foreign gene into one tube (+AMP) of the bacteria and then leaving another plate (-AMP) pure, this experiment should answer any questions pertaining to the transformation of bacteria. Diagram: Procedure: Samples of E. Coli are scrapped off a nutrient agar plate and suspended into two tubes containing a solution of calcium chloride that were marked either +AMP or -AMP.
Both tubes were incubated at 0 degrees Celsius for 15 minutes. Then Plasmid p AMP was added to the +AMP tube. After the addition of the p AMP, a brief shock at 42 degrees Celsius followed to both tubes. Then both tubes were cooled once more and had Luria broth agar and Luria broth agar with ampicillin added to each.
Finally, the plates were incubated again for 24 hours this time at 37 degrees Celsius. The next day both were checked for bacteria growth. Results and Discussion: 1. Transformed Cells (+AMP) Wild Type Cells (-AMP) LB/AMP: lawn no growth LB: individual colonies lawn Because the E. coli is given the ampicillin resistant gene, the bacteria will grow whether or not the plate contained ampicillin or not. The results are just as obvious if the situation were reversed.
If the E. coli did not contain the ampicillin resistant gene, the bacteria would not be able to grow. Finally the E. coli bacteria could also grow without the aid of the ampicillin resistant gene as long as ampicillin is not present that would inhibit its growth. 2. A. ) The +LB plate contains the resistant gene which is not the case with the -LB plate. However, both plates are capable of growing.
Because of the +LB s resistance to the ampicillin it is able to grow in the presence of the ampicillin. The -LB plate does not have ampicillin thus, it is not affected by not having the resistant gene and still grows. B. ) The -LB/AMP plate has no bacteria growth because the plate has ampicillin but does not have the gene that resists the ampicillin. The -LB which also does not have the gene present will grow, however, because there is no ampicillin present on the plate. C. ) The +LB/AMP will grow because it has the resistant gene and will not be inhibited by the ampicillin present on the plate. The -LB/AMP plate will not grow because it does not have the resistant gene and the ampicillin present on the plate will kill the bacteria.
D. ) Both these plates will grow because both have the resistant gene. But it is more probable that the +LB plate that does not have any ampicillin present will have more growth because it does not contain any ampicillin that would interfere with the growth of the bacteria. 3. (a) Total mass of p AMP used in step 5. Concentration x Volume = Mass... 005 x 10 = . 05 g (b) 250 L (step 1) + 250 L (step 9) = 500 L. (total volume suspension) 200 L (step 12) = volume suspension spread 250 / 500 = 2 / 5 L = fraction spread (c). 05 (a) x 2 / 5 (b) = . 2 (mass p AMP spread onto +LB/AMP plate) (d) 10 (number of colonies observed) /. 2 (c) = 50 5. 0 x 10 = scientific notation = transformation efficiency. 4. There are a few factors that might have influenced the transformation efficiency. The amount of bacteria that was originally placed into the tubes is a factor that might have influenced the calculations on growth.
There might have been less growth than it appeared to be. Temperature which was important in this experiment could have also been a efficiency factor. Because temperature is hard to be precise with (especially in the classroom), there is a possibility that it could have affected the transformation efficiency as well. Moreover, there are all sorts of bacteria's that could have been introduced by our hands, the air and the tubes used that are also efficiency factors. Conclusion: The initial hypothesis was supported by the results of this experiment.
Through the introduction of the Plasmid DNA the resistance gene for the ampicillin was added to the makeup of the bacteria. Because of this gene the bacteria was later on able to grow in the presence of ampicillin, which would have otherwise killed the bacteria.
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