The Transformation of Escherichia coli
With pGalTM

OBJECTIVE: To develop an understanding of bacterial transformation by plasmid DNA, and determine the transformation efficiency of a bacterial sample.
INTRODUCTION:
Transformation is an important biological mechanism for generating genetic diversity in the natural world. It is also one of the most used tools used in modern biotechnology. It has allowed for a lot of genetic engineering advances. Scientists can design and build DNA containing genes they want and then transfer these genes into bacterial cells that they can get to express those genes. This is a very basic technique that is used on a daily basis in a molecular biological laboratory. This is based on the natural function of a plasmid: to transfer genetic information vital to the survival of the bacteria. Transformation is an example of horizontal gene transfer. There are three mechanisms for horizontal gene transfer in bacteria. Conjugation is the transfer of DNA from one cell to another through conjugation pili. Transduction is the transfer of genes between bacterial cells using a bacteriophage (a type of virus). Transformation is the ability of cells to take up freely floating DNA found in the environment. Bacterial cells that are able to take up free-floating DNA from the environment are called competent cells. Bacteria are not always competent. When growth conditions are optimal, most bacteria cannot uptake DNA. In most cases, in response to some stimuli (such as stressor starvation), a cell can turn on the genes that allow it to become competent. In nature, as bacterial cells die, their cells are lysed, spilling their plasmid DNA into the environment. The DNA chromosome is broken up into chunks that float freely in the environment. A competent cell could take up one or more of the chunks. If a gene that is taken up is similar to a gene already found in the chromosome, the two DNA sequences will line up beside one another and then swap. The old DNA is replaced with the new one. This is called recombination, where genetic material is mixed and matched. In disease, this has resulted in organisms that are resistant to antibiotics. Escherichia coli is a popular organism for use with transformation experiments. By briefly shocking the E. coli bacteria with a plasmid environment, they will grow blue on the AMP/X-gal plate. Transformation efficiency can be determined according to the following equation:
Number of transformants final volume at recovery number of transformants μg of DNA volume plated per μg
MATERIALS:
The materials used for this experiment included microcentrifuge tubes (referred to as transformation tubes), CaCl2 (calcium chloride), Bacterial culture (E. coli on LB Source plate), sterile toothpicks, sterile inoculating loop, disposable pipettes, plasmid DNA (pGal), buffers (control and recovery), ice water bath (10 ̊C), hot water bath (37 ̊C and 42 ̊C), agar plates (X-Gal control, and AMP X-Gal).
PROCEDURE/OBSERVATIONS:
Using a sterile toothpick, 15 colonies were taken from the LB source plate, and mixed in the prepared “–DNA” tube, which contained 1.0ml ice cold CaCl2 solution. 0.5ml of this solution was placed in the “+DNA” tube, which contained 0.1ml of pGalTM. 0.1ml of the control buffer was also placed in the –DNA tube. Both tubes were placed in an ice bath for 10 minutes. They were then placed in a 42 ̊C water bath for 90 seconds. The tubes were immediately returned to the ice bath and incubated for 2 minutes. 0.5ml of the recovery broth was added to each tube, using a pipette. The tubes were gently flicked to mix the solution, and then placed in a 37 ̊C water bath for 30 minutes. The agar plates were labeled (X-Gal Control 1, AMP/X-Gal Control 2, and AMP/X-Gal/pGal). After the recovery period, 0.1ml of the solution in the –DNA tube was transferred to the Control plates. 0.1ml of the +DNA solution was placed on the remaining agar plate. Using separate inoculating loops, the DNA samples were spread all over the agar plates. The plates were stacked one on top of the other, and placed in a 37 ̊C bacterial incubation oven for one week.
RESULTS:

Figure 1 – The plates after incubating in a 37 ̊C incubation oven for one week
No bacteria grew on the Control plates. There were approximately 61 blue colonies that grew on the AMP/X-Gal plate.
61 transformants 0.5ml 2440 (2.440 x 103) 0.05 μg 0.25ml transformants per μg
The transformation efficiency is 2400 transformants per μg. CONCLUSION: No bacteria grew on the control plates probably due to the fact that because there was no plasmid DNA in the environment, the bacteria, although competent, had nothing so they did not grow. They did were not able to adapt to their new environment. The blue colonies were due to the genetically manipulated E. coli which made is possible for the bacteria to glow, which shows a resistance to Ampicillin due to bacterial transformation.
LITERATURE CITED:
Chacko, Sherin. (2015, January 11). Copy of Bacterial Transformation Lab. Web. 20 September 2015.
Hartsock Angela. Bacterial Transformation: Definition, Process & Applications. Web. 20 September 2015