Lab Report On The Effects Of Bacterial Transformation On E. Coli Bacterium
Bacterial transformation is a form of biotechnology. It involves manipulating organisms for products and other purposes such as medication. Over the years, biotechnology has undergone what can only be called, a scientific revolution. It has led to vaccines being made in large amounts, healthier foods, safe and affordable antibiotics and more. Biotechnology involves changing the proteins produced by bacteria by "giving it new instructions. " DNA contains genetic information and "instructions" for biological development. The proteins carry out the instructions because the DNA codes for them. Our host bacteria were Escherichia coli, commonly known as E. coli. It was used because it grows at a rapid rate. It's important to note that bacterial DNA is organized in a double-stranded, circular model. Bacteria also have a plasmid. A plasmid is a small, circular, self-replicating DNA molecule separate from he bacterial chromosome. The plasmid has genes that give it the ability to pass on beneficial traits to other bacteria. Some bacteria also have genes that code for antibiotic resistance. The purpose of this lab is to explore the effects of bacterial transformation on E. coli bacterium through procedures such as heat shock and incubation.
We inserted pBlu plasmid into E. coli, which is supposed to turn blue to show its presence. And we also inserted ampicillin. There were positive and negative controls for both variables to determine accuracy. Our groups' hypothesis was that the E. coli with and without plasmids that receive the broth would exhibit growth and turn a white color. Additionally, we predicted that the samples that receive ampicillin with the plasmid (positive control) will grow due to a resistance gene, and the sample without the plasmid (negative control) will die. The positive control samples with the plasmids that received broth, ampicillin and X-gal will turn blue and grow. The negative control, with no plasmid that received broth, ampicillin and X-gal will die because once again, it is not immune.
Methods
There were many steps involved in conducting this experiment to receive accurate results. Firstly, we had to focus on safety and put gloves on and keep them on to work with any lab materials, and take them off to work with the stopwatch and any other non-lab materials. The goal was to insert the plasmid inside of the bacteria. We used a sterile plastic inoculating loop to transfer isolated colonies of E. coli to the tube with plasmid. The cells were immersed in CaCl solution and vigorously spun.
The same was done in a tube without plasmid. Both tubes were placed in ice. Using the inoculating loop, we added plasmid DNA to the +plasmid tube and mixed. We returned it back to the ice and incubated the -plasmid and +plasmid tubes in ice water for 15 minutes. In the mean time, we labeled the media plates accordingly. The labeled plates can be seen in the data/results section. After the 15 minute incubation, we had to "heat shock" the cells by removing the tubes and immersing them in a 42 °C water bath for 90 seconds. We added Luria (LB) Broth to each tube. While they were immersed, we gently agitated the tubes to mix the LB with the cell suspension. The tubes then recovered for 10 minutes. We removed some cells from the tubes and spread them on the 5 plates in a back and forth shaking motion. The plates were wrapped, and placed upside down in a 37°C incubator because that is the optimal growing temperature for E. coli. We came in for the next lab about a week later, and surveyed the results.
Discussion
Our group was happy with our findings because the results came out as predicted. The plasmid gave the bacteria culture a resistance to the ampicillin, which is why all of the plates containing a plasmid exhibited growth no matter what was placed in the E. coli sample. However, the samples containing no plasmid only survived if there was no ampicillin in them, because they are not immune to the antibiotic. There is always a chance of error when performing an experiment. Many factors can influence the transformations success.
The process of "heat shock" is very delicate. If not done correctly, the plasmids may not enter the cells and undergo the transformation process. This would be evident at the end of the lab when the cells containing plasmids do not survive in the agar plate with ampicillin. When transferring the E. coli either from the starter plate to the tubes or from the tubes to the agar experimental plates, contamination can occur through the air. This contamination from the air could cause the bacteria to change and not give accurate results.
