Analysis of Escherichia Coli PGLO Transformation Process

Introduction

This lab was conducted to change the genotype and phenotype of a bacterium called Escherichia Coli (E. Coil). This bacterium is commonly used in various experiments and most often because of how fast it grows and easy to work with. Escherichia Coli (E. Coil) is common within animals and can be found in the lower intestine of the digestive tract that has a rod-shaped form. These orgasms grow rapidly within a temperature range around 37.4 degrees Celsius and can be easily regulated in a lab, especially with nutrition as they obtain energy from a multiple source and also in an environment that does depend on oxygen.

In this lab, we worked closely with plasmids which contains genes that are necessary for survival a can be passed from one bacterium to another in which we used pBlu. pBlu contains genes for Ampicillin an antibiotic resistance and also Beta-galactosidase which is an enzyme that converts X-Gal into Indo Blue. The purpose of this was to observe if this bacterium Escherichia Coli successfully obtained genetic material from the external source which we call transformation the uptake and expression of genetic material.

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Materials and Methods

In the process of transformation, we began by labeling each two which were +pGLO and -pGLO then placing them into the tube rack. We then transferred 250 micrometers of CaCl2 solution into both the +pGLO and -pGLO tubes by using a sterile transfer pipet. After sealing both tubes they were then placed into case that contained crushed ice on the bottom to control temperature.

Next, using a sterile loop, we picked up a colony of Escherichia Coli into the sterile plate, then using the +pGLO tube only, we dip the loop into the transformation solution. We then spin the loop between the entire colony was spread in the transformation solution.

The tube was then transferred back into the rack holder. We then repeated this process beginning with a new sterile loop but this time using the -pGLO label tube. We continued to the next phase which was obtaining a new sterile loop to dip into the plasmid DNA stock tube, we withdrew a loopful to mix into the +pGLO tube only. In this next phase we put both tubes of positive and negative pGLO in an incubator for 10 minutes as the tubes were sitting, we then label four of our agar plates as -pGLO (LB), -pGLO (LB/AMP), +pGLO (LB/AMP) and lastly +pGLO (LB/AMP/ARA). After all labeling was completed we started the process of using heat shock, we used the same rack holder to transfer both the positive and negative pGLO tubes into water with a set temperature of 42 degrees Celsius, the tubes were observed carefully that they were at the bottom in order for the bottom of the tubes to stick out and make contact warm water. After 50 seconds we took both tubes back on the ice for 2 minutes.

We proceed with the next phase that required us to remove the rack containing the tubes because we then needed to open a tube one of the positive and negative pGLO tubes and take a sterile pipet to transfer 250 micrometers of LB nutrient broth into the tube. This step was repeated using a new sterile pipet for the broth as this was extremely important to not cross contaminate the pipets. The tubes were then placed at room temperature for 10 minutes then proceeded to carefully flick the closed tubes with a finger to the solution. Using a new sterile pipet for both the positive and negative pGLO tubes we transferred 100 micrometers into the corresponding label plates. Then used another new sterile loop for each individual plate to allow to evenly spread out contexts within the agar. The final step of this required these plates to be stacked on top of each other sealed with tape on for 7 days.