Quick and easy bioengineering procedures: Competent cells and transformation

Hi all,

This is the second post in a series of 4 in which I want to share a quick and easy to use procedure for engineering a yeast strain. The idea is to go from the cloning stage, right after doing a PCR (or order) to get the DNA you want to clone, up to the process of transforming the yeast and selecting the positive clones. Keep in mind that the goal here is to make it quick and easy, so I'm targeting those who are not experimental biologists and want minimal lab work, or those who have a super wonderful, really cutting edge protocol, but maybe go overwhelmed in terms of time and resources and want to simplify the cloning and transformation stages of the project.

In this post I share the procedure for making competent E. coli cells and transforming them to take up plasmids, ideally generated following the procedure explained in the first post of this series (link). Nonetheless, in principle, these cells should be able to take up any foreign DNA. What I show here is known as chemically induced competence followed by heat shock transformation. In fact, there are more efficient transformation techniques than this, such as electroporation. But in my experience, heat shock transformation is faster because it allows multiple samples to be parallelized, it is much cheaper because you don't need fancy cuvettes, and it generally brings decent efficiency if the constructs you want to transform are not particularly complex. However, if you are dealing with a host that has specificities, or if for some of your constructs you are unsuccessful with the technique presented here, you might consider checking if the previous cloning steps are working, try pretreatments for capsids or cell wall, or try electroporation. Thus, this approach is convenient when you have several constructs to transform, they are not too complex, and you are dealing with a host that is not a weirdo.

The overall goal of this series of posts is to engineer a yeast strain, so we will use E. coli only as a carrier for the plasmids to propagate them. In this regard, an ideal carrier would increase the probability of transformation by minimizing nuclease activity and unwanted DNA recombination. One of my favorite strains for this is the E. coli strain called DH5-Alpha, this strain is quite widely used in molecular biology labs and you can easily find it just by asking on your institution's mailing list, otherwise you can buy it.

So, once we know which strain to use as a carrier, the first thing to do is to make the cells competent for transformation. Competent cells are nothing more than cells that have been treated to improve their ability to take up foreign DNA. The theory behind this is complicated if we get into the small details, but basically we will alter the permeability of the membrane and neutralize unwanted interactions of the DNA with the bacterial surface by adding ions. We will also add glycerol, which will allow us to store the cells at -80°C for long periods and use them whenever we need. An important detail of this procedure is that, once started, it is imperative to keep the cells always below 4°C. This restricts cell membrane fluidity, maintaining the enhanced permeability.

For transformation, we will mix the competent cells with the DNA we want to transform. In my case I always add the product of the Golden Gate reaction. You can always purify the DNA by dialysis or using commercial kits, but in my experience it is not necessary. Then, once the DNA and competent cells share space, keeping everything below 4°C, the mixture is exposed for a brief period to a heat shock at 42°C. Again, the theory is complicated, but in short the elevated temperature increases the Brownian movement of molecules out of the cells, thus increasing the probability that a DNA molecule will cross the cell membrane. After this process, the cells are recovered in rich media at 37 °C for two or three cell generations and plated on selective media.

After this introduction I want to give you some final notes that may be interesting. First, you can use the last tube of competent cells as a source to make more component cells. Second, you can scale up the protocol for competent cells to make larger batches, and store small amounts for individual reactions (or for 3 reactions to have a positive and negative control). Third, when transforming it is always good to add an extra reaction for the negative control, for example by adding water instead of DNA. This way, you make sure that your host cells do not grow in the selective medium, but do grow in the non-selective medium. It is also very convenient to transform the cells with some plasmid that serves as a positive control (e.g., the backbone of the construct) to check that the protocol works. Thus, cells transformed with the backbone should grow in selective media.

PROTOCOL FOR COMPETENT CELLS:

1. The night before, inoculate a 5 ml culture and grow overnight.
2. Next morning dilute cells ~ 1:200.
3. Grow the cells to an OD600 of 0.5 – 0.6 (This takes about 3 hours with DH5-Alpha)
4. Spin down the cells at 4 ºC, 4000 rpm, 15 minutes. Note: Keep the cells at 4 ºC from now on.
5. Resuspend cells in 15 ml, ice-cold 100 mM CaCl2.
6. Leave on ice 4 hours to overnight.
7. Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.
8. Resuspend cells in 4 ml, ice-cold 100 mM CaCl2 + 15% glycerol (final concetration).
9. Aliquot 70 µL of cells into pre-chilled tubes (70 µl for approx. three transformations).
10. Use immediately or store at -80ºC. Note: Frozen cells are only good once.

PROTOCOL FOR TRANSFORMATION

1. Thaw CaCl2 competent cells on ice.
2. Place 20 µL of cells in a pre-chilled tube.
3. Add 3µL of golden gate product (or pure DNA).
4. Mix gently by flicking the tube.
5. Chill on ice for 10 minutes.
6. Heat shock at 42 °C for 30 seconds.
7. Return to ice for 2 minutes.
8. Add 200µl LB medium and recover 60 minutes the cells by shaking at 37 °C.
9. Plate out the cells on selective LB, one plate with 20µL, the other one with the rest of volume, up to 200µL.
10. Incubate at 37 °C overnight.

The next morning, if you observe colonies with the expected phenotype for positives: CONGRATULATIONS!!!.

It's done, you should be ready for colony PCR to recheck and you may also want to sequence after a miniprep.

So, in post number 3 of this series I will share the procedure on how to perform yeast transformation with the plasmids constructed and obtained in post 1 and 2.

Good luck with the procedure, and I am available and happy to discuss any aspect of it.

Regards :)

REFERENCES:

Asif, A., Mohsin, H., Tanvir, R., & Rehman, Y. (2017). Revisiting the mechanisms involved in calcium chloride induced bacterial transformation. Frontiers in microbiology, 8, 2169.

Tu, A. H. T. (2008). Transformation of Escherichia coli made competent by calcium chloride protocol. American Society For Microbiology, 8(1), 1542-1546.