Quick and easy bioengineering procedures: Golden gate cloning

Hi all,

I have made this post as the first of a series of 4 in which I want to share a quick and easy procedures 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, all the way to the process of transforming the yeast and selecting the positive clones. Note that the goal here is to make it quick and easy, so I am 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 overkilling on time and resources, and want to simplify the cloning and transforming stages of the project.

(So, yes, it's me telling you: Don't worry, it's OK as long as you get the clones you wanted. The time you save in the lab is precious for reading papers. WOW!!!)

Nonetheless, if things go wrong with the procedure presented here, you might consider troubleshooting or finding another more specific protocol, but if you are not doing complicated constructs involving large chunks of DNA (my record with this protocol is cloning some inserts up to 5Kbp), these protocols should work fine. What I share here worked quite well for me to engineer many strains and generate hundreds of plasmids with three and even more transcriptional units (this is assembling around 8 parts in the golden gate jargon).

In this first post I am going to share the procedure for cloning and the detailed protocol for using modular cloning (MoClo) based on Golden Gate assembly. This approach is very convenient when you want to insert and shuffle several transcriptional units within the same backbone. The golden gate methodology is based on the use of type IIS restriction enzymes, as opposed to traditional restriction and ligation strategies, these enzymes cut outside of the target recognition sequence, generating cohesive ends on demand. Therefore, it allows multiplexed insertions in a single reaction, which incredibly increases the flexibility of cloning. In addition, MoClo, based on the stepwise cloning process, allows you to interchange different parts as desired by designating specific overhangs to each type of part. All of this may sound complicated, so I've included some references at the end of the post, and this resource to better understand the technique:

https://blog.addgene.org/plasmids-101-golden-gate-cloning

There are several preexisting libraries with predetermined parts and infinite extension possibilities. For yeast, the one I know best, and probably one of the most used is the Yeast Tool Kit (YTK). There are also preexisting libraries for bacteria and other organisms, and you can find them in the addgene repository.

Here come some of the limitations of the golden gate method: well, the restriction enzymes you use for cloning (usually 2 if you are doing MoClo) must be present only at the sites intended for cloning. So, if you are designing DNA on demand, keep this in mind. Otherwise, if you can't avoid having restriction sites in addition to those mentioned, you can simply do targeted mutagenesis or use Gibson assembly. The advantage of the golden gate over the Gibson assembly is that the former would allow you to clone shorter fragments of DNA, which could be convenient in some cases, especially if you make small home-made libraries.

Now, just to finish with the introduction, I want to stress some general aspects that have to take into account during the design stage. So when you are designing the backbones and the distribution of the transcriptional units try to do such that you (i) minimize the cloning reactions, (ii) minimize integrations, and (iii) are able to characterize the different modules in parallel and independently.**

PROTOCOL:

The first step is to prepare the DNA dilutions by bringing it to 20fmol (10^-15 moles), if possible in 1 µL of each plasmid.

Next, the reaction mixture is as follows:

REACTANT VOLUME/SAMPLE (ul) Water Up to 10 µL T4 ligase buffer (10x) 1 Restriction enzyme 0.8 Bovine Serum Albumin (100 µg/ml) 0.8 T4 ligase 0.4 Total 10

Note that in the same reaction volume you will do the restriction and ligation of all fragments, that is the point of golden gate cloning. I use T4 ligase buffer as the reaction buffer, and bovine serum albumin (BSA) is used to avoid nonspecific enzyme interactions, it really makes a difference in my experience.

The reaction mixture is introduced into the thermocycler with the following settings:

1. 37°C for 15 minutes (activation)
2. 37°C for 2 minutes (restriction)
3. 16°C for 5 minutes (ligation)
4. Back to step 2 x50 times (many restrictions and ligations, you can reduce the number of cycles according to the complexity of the reactions)
5. 37°C for 15 minutes (final restriction and ligations)
6. 50°C for 5 minutes (inactivation)
7. 80°C for 5 minutes (inactivation)

Once the reaction is done, it will be ready to transform and propagate, you can also store it at 4ºC.

So, in post number 2 of this series I will share the procedure on how to prepare thermocompetent cells for quick and easy transformation.

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

Regards :)

REFERENCES

Golden gate: Carola Engler, Romy Kandzia, S. M. (2008). A one pot, one step, precision cloning method with high throughput capability. PloS One, 3(11). https://doi.org/10.1371/JOURNAL.PONE.0003647

YTK: Lee, M. E., DeLoache, W. C., Cervantes, B., & Dueber, J. E. (2015). A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synthetic Biology, 4(9), 975–986. https://doi.org/10.1021/sb500366v