This shows the data that was acquired after the lab. The dish with the plasmid and Luria broth and ampicilin showed the growth of 13 colonies of bacteria. The plate with no plasmid and broth and ampicilin showed no growth of bacteria. The plate with broth and ampicilin showed an enormous amount of bacteria growth, which was too much to count. This is called a lawn. The last plate also had a lawn as there was no plasmid or ampicilin.
Calculating Transformation of Efficiency
Transformation efficiency is expressed as the number of antibiotic-resistant colonies per μg of plasmid DNA.
- 10 ul (microliter) of plasmid was used at a concentration of 0.005 ug/ul, so the first step would be 10 x 0.005. This equals 0.05.
- Calculate the total volume of cell suspension prepared. 10 ul of plasmid + 250 ul of Luria broth + 250 ul of CaCl2= 510 ul.
- Calculate the fraction of the total cell suspension that was spread on the plate. 100 ul of cell suspension / 510 ul= 10/51= 0.196
- Determine the mass of plasmid in the cell suspension spread. Total plasmid mass (#1) x fraction spread (#3)= 0.05 x 0.196= 0.0098
- Determine the number of colonies per μg plasmid DNA. Colonies observed / mass of plasmid (#4)= 13 / 0.098= 1326.53
Transformation efficiency= 132.65
Calculate the transformation efficiency when the amount of plasmid used was 20 ul at a concentration of 0.001 ug/ul and 200 ul of Luria broth and calcium chloride was used. Thirteen colonies were counted and 100 ul of cell suspension was used.
Natural Selection- Resistant bacteria grow in population by natural selection. Bacteria that are resistant to ampicilin will survive when ampicilin is present. The rest of the bacteria that do not have the adaptation to be resistant die. Therefore, the resistant bacteria pass on their genes and the offspring are resistant. Only the fittest survive, which exhibits natural selection.
Plasmids- Plasmids are small DNA molecules within a cell that are physically separated from a chromosomal DNA and can replicate independently. The plasmids in the lab have pGreen, which is a gene that makes the bacteria fluorescent under a UV light, and they have the ampicilin resistant gene so the bacteria will not die to the ampicilin in the agar.
Asexual Reproduction- Bacteria undergo asexual reproduction and the offspring are genetically identical to the parent. In this lab, when one bacteria that as the plasmid reproduces, more and more resistant bacteria are made. The plasmid is inserted into a couple bacteria and they reproduce, producing clones that are also resistant.
Answer to Practice Problem
- 20 ul x 0.001 ug/ul= 0.02
- 20 ul + 200 ul + 200 ul= 420
- 100 ul / 420 ul= 0.24
- 0.02 x 0.24= 0.0048
- 13 / 0.0048= 2708.33