DNA Cloning for Bacterial Protein Production
Once thought to be science fiction, DNA cloning has become a routine practice in protein research labs. Since the production of proteins for scientific study is a fairly difficult and pricey process, DNA cloning using a unique strain of E. coli allows for targeted protein production using the bacteria’s already optimized cellular machinery. [5]
The process of DNA cloning is well refined and best thought of in the following steps [4]:
- Preparation of an insert. This is a small DNA segment that ultimately codes for a scientist’s desired protein.
- Preparation of a plasmid vector. This is what will carry the insert DNA into the bacteria, while also providing a few other functions necessary for DNA cloning. [2]
- Joining of the insert and plasmid vector in a process referred to as ligation. Once joined, the insert and plasmid vector pair is now called a recombinant plasmid.
- Introducing the newly joined recombinant plasmid into the E. coli bacteria.
- Confirming that successful E. coli clones have been made using techniques like colony PCR and DNA sequencing.
There are two methods of DNA cloning that are most common in research labs. They are restriction enzyme cloning and ligation independent cloning (LIC). While both methods follow the same general steps listed above, they differ in the joining/ligation step. Specifically, the LIC technique does not require help from an enzyme to join the insert and plasmid vector together, while restriction enzyme cloning does. [1]
Insert Preparation
As previously mentioned, cloning begins with an insert. Also referred to as the gene of interest or target gene, this is the DNA segment that will be inserted into the plasmid vector and ultimately codes for a scientist’s target protein. These inserts are usually ordered directly from companies that specialize in the generation of high quality DNA fragments. Once received, the amount of delivered insert needs to be increased to allow for efficient cloning. [6] To do this, a lab technique called PCR is used. PCR stands for polymerase chain reaction and it lets scientists make several copies of or “amplify” a DNA segment. [4] To learn more about PCR, one of the foundational techniques of DNA cloning, click here. Following the PCR amplification, the insert DNA will need one more modification before it’s ready to be joined with a plasmid vector. The purpose of this final modification is to generate regions on the insert DNA that will allow it to be attached to the plasmid vector. The way this modification is done varies depending on the cloning technique being used (restriction enzyme cloning or LIC), and is what leads to the differences in the ligation step between the two techniques.
Vector
There are many options to choose from when it comes to picking a plasmid vector, as many have been specifically engineered for the purpose of cloning. [2] Despite the variety of options, most plasmid vectors contain little more than what is necessary for DNA cloning. They commonly include segments for antibiotic resistance, replication start sites, etc. [2] Similar to insert preparation, plasmid vectors are typically ordered directly from companies that specialize in their synthesis. A scientist can also create their own plasmid that can be completely customized to meet their specific lab needs. Regardless of using a predesigned or custom vector, a plasmid can actually be reused with multiple inserts by simply “cutting out” the previous one.
Conveniently, the preparation of a plasmid vector for both restriction enzyme cloning and LIC is just about identical to that of insert preparation, as both techniques create regions that allow for the plasmid vector to be attached to the previously treated insert. It is worth noting that the same technique must be used for both an insert and plasmid vector if they are to be joined properly.
Ligation
The ligation, or joining, step is where the prepared insert and plasmid vector are joined together at their specialized attachment regions. This is also the most distinguishing step between restriction enzyme cloning and LIC due to the slight variations in insert and plasmid vector preparation between the two techniques. For restriction enzyme cloning, the insert and plasmid vector are mixed together with an enzyme called DNA ligase and incubated anywhere from a few hours to a full day depending on the temperature. For ligation independent cloning, instead of adding the DNA ligase enzyme, the insert and plasmid vector are mixed together and left at room temperature for a short incubation of 5–10 minutes. [3]
Transformation (We’ve got clones!)
Now that the insert and plasmid vector are joined together to make a recombinant plasmid, the next step is to introduce the DNA into the E.coli bacteria through a process called transformation. Transformation is simply the mixing of E.coli bacterial cells with the recombinant plasmid and introducing a sharp temperature shock that should trigger the bacterial cells to take up the plasmid. [5] After a brief growing period for the cells, they are then spread on an antibiotic treated agar plate and allowed to grow overnight. The purpose of using an antibiotic treated plate is because the plasmid vector has a region that allows for cells that carry it to be resistant to the antibiotic. This means that only bacteria cells with the recombinant plasmid should grow on the agar plate. If everything worked correctly, the plate should have bacterial colonies made up entirely of genetically identical clones!
Finding positive clones
While the agar plate may be full of bacterial colonies, it is important to make sure and confirm that the colonies on the plate are ones that have successfully taken up the full recombinant plasmid. This is where a technique such as colony PCR (CPCR) comes in handy. The principle behind CPCR is amplifying a small segment of the DNA inside the bacterial cells that make up a single bacterial colony. [7] This DNA amplification can then be used to see if the recombinant plasmid is present inside the bacterial cells of that colony. A DNA extraction can also be performed on a bacterial colony and sequenced to ensure it’s DNA is correct and free of mutations.
Downstream
Once a positive colony has been found, a scientist now has a bunch of successful DNA clones! These clones can now be used in downstream protein production. This involves growing a large batch of the cloned bacteria and triggering the cells to start producing proteins. From there, a purification process is done to retrieve the proteins that have been produced, which scientists can now use in further lab studies.
Conclusion
Here at Macromoltek, we utilize a similar cloning process to produce some of our own custom proteins. While perhaps not as exciting as fully cloned sheep or humans, DNA cloning in bacteria has and will continue to afford scientific breakthroughs that were once thought to be unattainable.
Citations:
1. Biolabs, N. (n.d.). Foundations of molecular cloning — past, present and future. Retrieved April 23, 2021, from https://www.neb.com/tools-and-resources/feature-articles/foundations-of-molecular-cloning-past-present-and-future
2. Lodish, H. (1970, January 01). DNA cloning with plasmid vectors. Retrieved April 23, 2021, from https://www.ncbi.nlm.nih.gov/books/NBK21498/
3. München, H. (n.d.). Cloning. Retrieved April 23, 2021, from https://www.helmholtz-muenchen.de/pepf/protocols/cloning/index.html
4. Nature news. (n.d.). Retrieved April 27, 2021, from https://www.nature.com/scitable/definition/polymerase-chain-reaction-pcr-110/
5. Overview: Dna cloning (article). (n.d.). Retrieved April 23, 2021, from https://www.khanacademy.org/science/ap-biology/gene-expression-and-regulation/biotechnology/a/overview-dna-cloning
6. Polymerase chain reaction (pcr) fact sheet. (n.d.). Retrieved April 23, 2021, from https://www.genome.gov/about-genomics/fact-sheets/Polymerase-Chain-Reaction-Fact-Sheet
7. What is colony pcr? (2020, January 10). Retrieved April 23, 2021, from https://geneticeducation.co.in/what-is-colony-pcr/
8. Khan Academy (2021, April 29) http://www.khanacademy.org/. Note: All Khan Academy content is available for free at www.khanacademy.org
