Why did our competency control have no bacterial colonies?

Because this was probably your first time handling competent cells and they’re sensitive to temperature changes and rough treatment. Mishandling the competency control will kill all the cells in that reaction, leading to 0 colonies and a false negative.

Transformation: How does heat shock work? What is the mechanism?

Briefly, the heat pulse helps release lipids that are on the membrane, which opens up pores in the membrane and lowers the membrane potential because of the gross change in membrane permeability. The rapid removal of lipids opens up a physical space in the membrane that DNA can pass through. Membrane potential is formed by the difference in electrical potential between the interior and exterior of the cell (look it up if you haven’t heard about it before… it’s super cool). Altering the membrane potential helps put the negatively charged DNA in close physical contact with the membrane and pull it through the holes by attraction. https://www.ncbi.nlm.nih.gov/pubmed/18651316

How quickly does a bacterium “heal” or “repair” after heatshock or electroporation?

The short answer is that it depends on the extent of the damage and the specific properties of that bacteria’s membrane. The recovery rate is a function of how long it takes the membrane to shrink or stretch to fill the holes or and how long it takes to restore the resting potential.

Could you further explain controls in the ligation experiment? I’m still not entirely sure what a control for background does – can you explain it again?

I’ll go over them again as review at the beginning of the next lecture. Briefly, controls are conditions you include to give you evidence that your assay is working and to help you figure out what went wrong if it did not work.

Postive control = any condition that you expect to produce a positive result in your assay

Negative control = any condition that you expect to produce a negative result in your assay

Competency control = do my competent cells work? positive control

Ligation control = does my ligase enzyme function?  positive control

Control for background, ligase + vector with no insert = can my vector re-ligate? negative control

No ligase control = is there anything in my ligation reaction components that can be transformed and convey antibiotic resistance? negative control

No DNA control = Do my antibiotic plates function properly? negative control