Bacterial Transformation Using Competent Cells
Bacterial cells were made competent and transformed to have ampicillin resistance and fluorescence
This experiment focuses on bacterial transformation using competent cells. To make competent cells, they must be soaked in a CaCl solution and heat shocked to allow the DNA to enter the cell. Once the cell is competent, the pSKII plasmid is added to allow ampicillin resistance. These transformed cells are plated on media with ampicillin, along with a few controls. In this experiment, it is assumed that the enzymes, likely KpnI, didn’t cut correctly and made the plasmid linear
Needs more content here, like some information on the operon we were cloning, the phenotype, and a brief mention of the results that led us to believe one of our enzymes was off. 7/10
Bacterial transformation is a technique used to allow cells to take up foreign DNA. In order to create competent cells, the cells need to be treated with a CaCl2 solution and heated fpr a brief period to allow them to take up the DNA. The CaCl2 solution puts holes in the cell membrane for the DNA to get in, and the heat shock allows the cell to physically take up the DNA.
For this experiment, the chosen cells were XL-1 Blue cells. These cells were grown in Luria-Burtani broth (LB) in the shaker overnight to allow the cells to grow at a rapid rate. This method allowed the cells to be used during the log phase, where the number of cells is doubling. Having a high concentration of cells is important because the more competent cells you have, the more transformed cells you have. To transform the competent cells, the DNA needs to be thawed and the pSKII plasmid DNA needs to be added. A CaCl2/glycerol solution should be added to the cells to prevent crystallization, and they are incubated to allow the DNA to adhere to the cell membrane. Then, the cells are heat shocked to allow the DNA to go inside the cell. Then, a rich growth medium is added, and the cells get gently shaken to allow the cells to recover and express the antibiotic resistance from the plasmid. Why do we care about antibiotic resistance here--i.e. how does this help us?
The pSKII plasmid is a polylinker that has a multiple cloning site that allows foreign DNA to be inserted without disturbing the plasmid. The two enzymes that will be used to cut the plasmid are KpnI and EcoRI. This plasmid also has ampicillin resistance, which is why the plates used in this experiment are LB-AMP-IPTG-XGAL plates. IPTG is an analog of lactose and induces transcription of the lac operon, which means that the cell can’t stop transcription and the operon is always on. XGAL is there to allow the cells to have a blue growth and will indicate if the lacZ gene is being expressed.
For the competent cell transformation, there are two controls being done. One is the transformation efficiency control, where the colonies were counted on the plates and the transformants per µg of DNA was calculated to ensure that there were enough transformed cells to allow the next part of the experiment to succeed. The other control is the selection control, which was used to determine that the cells would actually grow on medium containing ampicillin and that the cells have transformed DNA.
The controls for the lux operon experiment are the background control and ligation efficiency control. The background control took the digested vector DNA and plated it along with the experimental. Ideally, this would result in little to no growth, which would show that all the DNA was digested. If the colonies are blue, then either one or both of the enzymes failed to digest, or the vector didn’t cut. (should be no colonies; linear plasmid gets degraded usually) The ligation efficiency control is to determine if ligation didn’t occur. This would result in no growth on the plates, and can be caused by either bad buffer, bad ligase, or a poor vector. For this control, both restriction enzymes need to be heat inactivated to prevent them from cutting the ligated DNA. Then, a mixture of water, buffer, and DNA enzyme will be added, and the solution will be incubated overnight. This will allow the ligase to re-insert the stuffer fragment that was removed to allow the new DNA in. This regenerates the plasmid vector and retransforms the cells. Ideally, there should be many blue colonies on the plates. If there is no growth, then the ligation was unsuccessful. If there is white growth, the vector wasn’t digested properly, and the plasmid was rearranged. 9.5/10
To begin, the culture was grown overnight in LB broth with 12.5µg/mL tetracycline, then 50mL of LB broth was inoculated with 1.0mL of the culture. The flask was shaken at 37ºC for a few hours to allow the cells to grow, and the optical density (OD) was measured. Once the OD hit 0