Bacterial Transformation via Electroporation

In this lab we shocked cells to cause their membranes to open up and absorb the surrounding plasmids. reference.

Vocabulary:

Transformation: "Transformation is the process that occurs when a cell ingests foreign DNA from its surroundings."

electroporation: "The action or process of introducing DNA or chromosomes into bacteria or other cells using a pulse of electricity to briefly open the pores in the cell membranes."

DIY version of an electroporator

Heat shock"Heat shock transformation uses a calcium rich environment provided by calcium chloride to counteract the electrostatic repulsion between the plasmid DNA and bacterial cellular membrane. A sudden increase in temperature creates pores in the plasma membrane of the bacteria and allows for plasmid DNA to enter the bacterial cell."

cuvette: "a straight-sided, optically clear container for holding liquid samples in a spectrophotometer or other instrument." We used a cuvette for electroporation.

glass beads: Used to spead a bacteria cultures evenly on a petri dish.

Enzyme: "A substance produced by a living organism which acts as a catalyst to bring about a specific biochemical reaction."

ROP protein: (repressor of primer). a protein that regulates replication.

kissing complex: "Kissing" is refereing to RNA that folds back on itself and binds to a section of opposite bases. Two kissing stem loops can also interact, called loop-loop pseudoknots. These interaction are responsible for forming tertiary or quaternary structure of many RNAs.

Origin of replication: a particular sequence in a genome at which replication is initiated.

competent cells: a cell that is capable of taking up exogenous DNA from its surrounding environment.

Lab notes:

This week we electroporated cells so that they could absorb surrounding plasmids. We pipetted a mixture of glycol, the cells and the plasmids and dropped a sample on a cuvette to be electroporated, and thus go through bacterial transformation. A lot of this week was focused on hardware development, thinking about what tools synthetic biologist need and ways to design them such that they are more acessible.

This is a link that describes guidelines for optimization of the electric puls in electropation.

Below are pictures of a standard electroporator. It's pretty simple hardware, just plastic injection molded pieces, however the cuvettes cost $3 each, and you need one for each sample. The electronic hardware is really the critical component for this system though. For bacterial transformation cells need to receive 1,700V for 5ms. The picture of the oscilloscope screen shows the actual waveform measured from a BioRad electroporation pulse generator. I was surprised to see it is just the exponential decay of capacitive discharge and one of the BioRad pulse generators costs around $3k.

I'm curious what the optimal waveform is to cause the cells membranes to open up. Was capacitive discharge just the easiest thing? Or is there a physics explaination to it?

We plated the sample onto petri dishes Noah prepared with an anti-bacterial, ampicillin. The plasmid we tried to introduce was pBlu which expresses a resistance to ampicillin, thus, if the cells did not transform they would die on the petri dish, but if they did take in the plasmid they would grow. Unfortunately, none of our cells transfomred because we were using experimental hardware for the electroporation. Noah preformed the same experiment using heat shock instead of electroporation
Noah preformed the same experiment using heat shock instead of electroporation however and demonstrated cell growth. He took this beautiful image.

After ~5 days we found that some of the student cells did acheive transformation and survived! Wow!

I'm super curious about the physics that govern the efficiency of electroporation causing the cell membrane to open up and if there's a way for the hardware to tailor its output wayform depending on the cells and sample properties. This was an interesting reference paper:
J.-M. Escoffre, T. Portet, L. Wasungu, J. Teissié, D. Dean, and M.-P. Rols, “What is (still not) known of the mechanism by which electroporation mediates gene transfer and expression in cells and tissues,” Mol. Biotechnol., vol. 41, no. 3, pp. 286–295, Mar. 2009.

Questions

Q. What genes are encoded by the pBlu plasmid?
See Carolina
A. pBlu "contains gene for ampicillin resistance and the entire ß-galactosidase gene."

Q. Given the LB-agar plates were prepared with ampicillin and a compound like x-gal, what type of confirmation do we have for whether plated cells were transformed with the pBlu plasmid?
See β-lactam antibiotic
See X-gal
A. Because the petri dishes had ambicillin, and the pBlu plasmid contains a gene for ampicillin resistance, we will only see cell growth if the E. Coli transformed and absorbed the plasmid. In our case we know our electroporation didn't work because no ones cells grew.

Q. (bonus)How would you design an experiment to determine the transformation's efficiency?
See Colony-forming unit
A. "Transformation efficiency is defined as the number of colony forming units (cfu) which would be produced by transforming 1 µg of plasmid into a given volume of competent cells." You can ditute samples to make a series of plates that have different concentrations of cells, and one should have a good number of colonies to count. Not too many colonies so that the plate is covered, or to few that it's sparse and not statistically significant.