Abstract

DNA is the foundation of life sciences. For us to be able to extract purify and then manipulate it is key to our advancement in this field. The vast benefits of such technology outweigh the delicacy and long process that is involved to make sure everything is done right. Specific volumes, chemicals and time, go into this process. Each step is tailor made to its purpose and the ultimate aim is to identify and replicate the required gene of interest, that will benefit us.

Aims and Introduction

Genetic engineering has evolved from the quest for human DNA mapping to vast other branches, ranging from cloning to artificial selection processes. The benefits have been laid out from the onset, with cures for disease and an end to genetic disorders amongst the more highly rated ones. However, each experiment has to be carried out accurately and safely in the laboratories before human testing can begin, hence the use of the bacteria, E Coli. There are numerous reasons for using E Coli for such experiments. I will highlight but 2 of them that are related to this report. First is the fact that the plasmid DNA (puc18) in E Coli is a high copy number plasmid, i.e. about 20-30ug of plasmid DNA obtained as compared to 3-10ug from low copy number plasmids . The second reason is there is a presence of multiple cloning sites (or MCS, which enable any gene of interest to be put into the carrier) in E Coli. Transformation is the process whereby a host cell takes up foreign plasmid DNA. These cells are known as competent cells. Transformed cells are the type that I hope to isolate during the course of this experiment. Although the process of transformation is a long one, involving numerous precise steps and reagents, the benefits are limitless, since transformation is a keystone step in any genetic engineering process. The goal of this experiment is to extract purified plasmid DNA for the purpose of transformation and cultivation. The secondary purpose is to be able to successfully isolate and identity transformed colonies. The role that experiments such as these are crucial if genetics is to become a key player in the life sciences field. As mentioned before, prevention of disease, tailor-made organisms as well as increasing genetic diversity1 are things worth investing in.

Materials and Methods Part 1:

The extraction of plasmid DNA from E Coli was done using the High Speed Plasmid Mini Kit (HSPMK), which enables us to isolate the plasmid DNA from the 1.5ml of E Coli culture used. The first step is to centrifuge the E Coli culture for 60 seconds and discarding the supernatant, and treating with 200ul of PD1 Buffer (50mM Tris-HCl pH 8.0, 10mM EDTA, 10ug/ml RnaseA). This allows us to obtain a clearer cell lysate with minimal genomic contaminants. This is known as the re-suspension phase. The next step is the lysis phase, where 200ul of PD2 buffer (200mM NaOH, 1% SDS (w/v)) is added to breakdown the lipids in the cell membranes. This is a fragile step, and caution must be taken during tube inversion to avoid shearing of the genomic DNA. The next step involves the addition of PD3 buffer (guanidinium chloride & acetic acid), which results in a lowering of the overall pH and neutralization, again, care must be taken to avoid shearing during inversion. After 10 inversions, the tube is centrifuged for 180 seconds. However, the supernatant obtained here is put through the PD column of the HSPMK. All flow-throughs are discarded. The next 3 steps involve a certain degree of washing, to remove any remaining contaminants. The first wash is by 400ul of W1 Buffer (guanidine hydrochloride & isopropanol). This buffer causes the residual proteins to get completely denatured. After another round of centrifuging, the contents are washed off with a 70% ethanl Buffer and eluted with 10mM Tris HCl at pH 8.5 (250C). The end product is purified plasmid DNA, which is used for transformation. 1ml of competent E Coli cells are added to 3 tubes containing 10ul of double distilled water and 5ul and 15ul of plasmid DNA. All 3 tubes are iced for 30min (to allow Ca2+ from CaCl2 to make DNA attach to the surface of the membranes), before being subjected to 90seconds of heat shock, which causes the DNA to be taken up into the cells. 1ml of LB broth is added to each tube, which facilitates the recovery phase for the cells. A 10-1 dilution of the transformed cells is done to have basis for comparison, and all samples of cells are spread on both LB and LBA plates.

Part 2:
2 different E Coli strains are grown in 4 different growth cultures. The strain of interest in this case is pUC18, which is ampillicin resistant and able to utilize lactose. Each petri dish contains a different growth medium, LB, LBA, LB+Xgal and MacConkey. Once single colonies are isolated, they are incubated onto gridded plates for further growth and easy identification. The next step is replica plating, where the end results is the

identification of the type of cells based on the characteristics of the colonies seen on each plate.

Results
Plates E Coli pUC18 LB Present Present LBA Absent Present LB+Xgal+IPTG White Colonies Blue Colonies MacConkey Pale red Colonies Bright red Colonies Table 1.1 Observation of growth of colonies on replica plating.

No. Of Colonies Plasmid DNA Type of Type of Plate added Suspension Positive Neat LB control Negative Neat LBA control 5ul Neat LBA 5ul 10x dilution LBA 15ul Neat LBA 15ul 10x dilution LBA Key: TNTC = Too numerous to count Table 1.2 Transformed colonies

Actual TNTC 12 TNTC TNTC TNTC TNTC

Relative 1 6 2 5 3 4

Discussion
There were no wrong results for the replica plating segment. Both E Coli and pUC18 showed colony growth on LB, since there was no ampicillin to eliminate the cells that did not contain the resistance gene. On the plate that contained ampicillin (LBA), the E Coli cells could not grow because ampicillin degrades the enzyme trnaspeptidase, disrupting the formation of the bacterial cell wall. However, pUC18 cells can grow because they have the enzyme that degrades ampillicin. In the Xgal plates, when the LacZ gene is disrupted, no beta-galactosidase will not be produced, hence the colonies produced will be white, since Xgal is not utilized. On the MacConkey plates, the pale red colonies seen are due to the E Coli converting lactose to lactic acid, hence lowering the pH. From the transformation results, it would be expected that all the negative control would have no colonies, since it only contained water and cells, and the 15ul plates would have more colonies than the 5ul plates. However, the negative control shows 12 colonies, probably due to

contamination of the competent cells or the plate with ampillicin resistant strains. The other anomaly is the 5ul neat plate having more colonies, relatively, as compared to the 15ul neat plate. This might have been a result of improper techniques, inaccurate measurement of volumes, or contamination.

References
1. http://www.biology-online.org/2/13_genetic_engineering.htm