Today we continue where we left off at the end of last week’s workshop. After the appropriate primers were designed by our capable participants and the DNA fragments amplified by PCR, it was time to assemble the fragments in a Gibson assembly reaction! We combined all the components we made in one reaction tube and added the Gibson master mix to allow it to work its magic, creating our desired genetic construct in a plasmid.
Afterwards, we transformed the plasmid into E. coli cells. Transformation is a process in which bacteria take up DNA from their environment. You could imagine that if you’re an E. coli cell happily swimming along in your environment, there would be no reason for you to take up random DNA molecules that might be toxic for you. So in the lab, we do two things to make sure transformation happens. First, we choose the DH5-alpha strain of E. coli, termed ‘competent cells’, meaning that they are specifically engineered to easily uptake DNA. Second, we heat-shock the cells, putting them in a water bath at 42 degrees Celcius for 30 seconds to induce an unfavourable environment for the cells, coaxing them into uptaking our plasmid.
By popular request, we looked at the results of our PCR last week. We presented an alternative method to check our results: by measuring how much DNA was produced. We used a spectrophotometer, which passes a wavelength of light through a DNA sample and measures how much DNA is there based on the intensity of the light that the DNA emits in response (absorbance). In our investigation, we added SYBR safe, a dye that intercalates DNA, which we know is excited at 280 nm and 520 nm. Again, we made sure to use controls: we showed that the least absorbance was with using water, followed by the dye alone, and then the dye combined with nucleotides and the template plasmid. Luckily, most of the PCR reactions last week were deemed successful as the absorbance was greater than if we measured the absorbance with the template plasmid plus nucleotides – suggesting that the DNA was indeed amplified!