Cloned Cats, Gibson Assembly and Gene Editing!
Meet Garlic, the world’s first “copy-cat”.
Why is she called a “copy”-cat, you might ask? That’s because Garlic wasn’t born through natural means. Instead, she was cloned from the cells of a DEAD PET. 🤯(whose name was — you guessed it-Garlic!)
THIS IS INSANE!!! All of you pet owners out there, you potentially don’t have to ever say goodbye to your favorite furry friend- you can keep making more of them!
With this kind of technology, we could theoretically clone any type of animal whose cells we can still find (so unfortunately no dinosaurs or dodo birds roaming around). Pretty soon, we’ll restore endangered species and bring balance to ecosystems!
How in the world is this possible? To find out, we’re going to dive into the world of molecular cloning.
What is molecular cloning?
Cloning is making a copy of an organism that has exactly the same genes. You might be thinking. “This sounds easy! Cloning just means we could point a tool at a cell, zap it, and we would have a completely different cell, right?”
Well…not exactly. When we talk about molecular cloning, we’re really just inserting a copy of a gene (vs. the whole genome of an organism) into a bacterial cell.
In the lab, molecular cloning is used to assemble recombinant DNA molecules. Recombinant DNA molecules are made from the joining of multiple DNA fragments.
How we can make Recombinant DNA
First, we need restriction enzymes. These enzymes are like a pair of scissors — they cut DNA at two specific sites in order to produce a DNA fragment. The cool thing is that they can cut up another organism’s DNA without damaging their own DNA!!
Cutting DNA with restriction enzymes produces strands with overhangs. To join these overhangs together, we need to have DNA ligase.
DNA ligase is an enzyme that’s like glue — it joins any two fragments of DNA together by creating bonds between the deoxyribose, which is a type of sugar that backbones of the DNA molecules consist of, between those fragments. This can be done for multiple DNA fragments of different organisms — creating our recombinant DNA.
Now that we have our recombinant DNA, let’s make a cloning vector!
But what is a plasmid? Plasmids are circular pieces of double stranded bacterial DNA that exist separately from the rest of a bacteria’s genome. Plasmid DNA + recombinant DNA = cloning vector!
But how exactly do we make a cloning vector? It’s simple — we take the gene of interest and insert it into the plasmid’s genome.
….
Got you there, didn’t I? As with many things in life, it isn’t so easy.
When you insert a gene into a plasmid, you have to be mindful of where you put it. Let’s take a look at a diagram of a plasmid.
The target gene is placed after a promoter, a genetic compound that’s like a light switch: in order to express your gene or light the room, you need to turn that switch on. A plasmid naturally contains a region where outside DNA sequences can be attached — here is where your gene sequence should be inserted. Tools like CRISPR-Cas9 are extremely helpful in this insertion.
We also need to insert a Kozak sequence close to our sequence of interest. Kozak sequences are like the current of electricity — this sequence will initiate translation of mRNA, which is the first step in the protein-making process.
Plasmids come equipped with a replication origin: the place where the plasmid DNA replication begins. Replication — like a copy machine — makes multiple copies of the plasmid’s DNA.
Replication continues around the circular structure of the DNA, making it easier for the inserted gene to be incorporated into the plasmid’s genome => This is the BIG THING for molecular cloning!
Cool, we have our plasmid with our recombinant DNA of interest. How can we put it into bacteria and observe what it does?
BACTERIA TIME!
TRANSFORMATION:
Bacteria are ingested with a plasmid and then heat shocked into taking the foreign DNA.
Heat shock allows pores in the cell membrane to open — just like how taking a hot shower seemingly opens up the pores in your skin, which allows dirt to collect and lead to any teenager’s worst enemy — acne.
You might be surprised to find out that lots of plasmids contain an antibiotic resistant gene. This is SUPER important, because when we want to figure out how many of the bacterial cells actually took up the plasmid (and contain our DNA), we can put the bacteria on an antibiotic plate to see which ones took the plasmid and DNA in!
The bacteria’s one part — how do we make sure the plasmid still has our DNA? Polymerase chain reaction (PCR) allows us to amplify and create multiple copies of the plasmid to check if the plasmid still has the DNA.
PROTEIN PRODUCTION:
If a plasmid contains the right control sequences, bacteria can be induced to express the gene they contain when a chemical signal is added.
The gene is then translated into mRNA which is then transcribed to make the protein.
Protein purification is the process of separating and isolating the protein from the rest of the plasmid.
What are some commonly used Molecular Cloning Methods?
- TOPO cloning: cloning without needing DNA ligase
- Gateway cloning: has a specific recombination DNA to be inserted to specific vectors
- And Gibson assembly!
What is Gibson assembly?
The Gibson assembly is a cloning method that allows for joining of multiple DNA fragments to be (mostly) seamlessly inserted into a plasmid.
How the Gibson Assembly works:
- The exonuclease chews back DNA from the 5' end. This inhibits polymerase activity and allowing the reaction to occur in one single process. The resulting single-stranded regions on adjacent DNA fragments can cool.
- The DNA polymerase (which generates nucleotides based on existing DNA fragments) incorporates nucleotides to fill in any gaps.
- The DNA ligase covalently joins the DNA of adjacent segments, thereby removing any nicks in the DNA.
- You can then go through the process of molecular cloning!
Why Gibson Assembly is one of the best cloning methods:
Cloning methods like Gateway cloning can only be applied to specific vectors and require the recombinant DNA fragments “att L1” and “att L2”.
But the Gibson assembly doesn’t require specific recombinant DNA — you can do it with any type of recombinant DNA!
How can cloning be used with CRISPR-Cas9 and in gene editing experiments?
Research has been done to show that CRISPR-Cas9 can be used as a restriction enzyme to cut DNA inside of a cell taken out of an organism (known as in vitro) for cloning with the Gibson assembly process. (if you didn’t catch the link to this before, you can read my article on CRISPR/Cas9 here!).
A small recap from that article: CRISPR-Cas9 uses the technique that bacteria use to defend themselves against viruses in order to delete, mutate or add genes. The insertion of genes is especially tricky, because the process needs to be precise.
Let’s see how we can increase that precision with the Gibson Assembly!
How it works:
- sgRNA (a combination of guideRNA and tracerRNA) was synthesized through PCR from a plasmid vector called pX330.
- It then underwent a purification process, and then the purified sgRNA and Cas9 nuclease were mixed together to form the Cas9/sgRNA complex.
- sgRNA led the Cas9 to the targeted sequence of the bacterial cell and Cas9 cut the double-stranded DNA at the target sites
- Gibson assembly process (as described above) was then used to insert the DNA fragment of interest.
Through doing their experiment, the researchers found two key things:
- The combined edits were MUCH more precise than using CRISPR-Cas9 by itself.
- Combining these two tools was more efficient: it took them 5 days to do total!
Gibson CRISPR-Cas9 as an experimental control:
Green fluorescent proteins are proteins that make an organism glow green. They’re just one type of protein used for experimental controls.
Gibson CRISPR-Cas9 can be used to insert sequences that code for GFP’s. These can then be used as controls for gel electrophoresis, a visualization of PCR,in comparison to performing gel electrophoresis assays for the gene of interest.
From making the most precise additions to serving as a way to create experimental controls, the Gibson assembly + CRISPR-Cas9 can be used to REVOLUTIONIZE the way gene editing is done!
Looking At A Broader Scale:
Going back to our friend Garlic the cat, we got a copy of the original that was almost completely the same through molecular cloning. CRISPR-Cas9, though, could insert genes to improve a new pet — we could insert a gene that would provide a natural resistance to whatever disease killed your pet in the first place!
Gibson Assembly was used to recreate the ENTIRE GENOME of the M. mycodes bacterium.
Gibson Assembly can turn DNA sequences into genes which code for chemical pathways. By observing what these genes do and how we can indirectly join them together along with CRISPR-Cas9 to make new compounds, we can do so many things. From creating new biofuels, to creating new biological systems to increase drug efficacy: the possibilities are endless!
Who knows? With Gibson Assembly and CRISPR-Cas9, you might say hello to a pet you once said goodbye too — but new and improved!
Want to see the AWESOMENESS of the Gibson Assembly in action? Stay tuned to see my presentation on how I simulated Gibson Assembly!
KEY TAKEAWAYS:
- Cloning is the process of inserting a gene into a bacterial cell by inserting a compound called a plasmid- molecular cloning looks at recombinant DNA specifically.
- Recombinant DNA is made of joining multiple DNA fragments together — like creating a SUPER gene!
- When you insert the recombinant DNA into the plasmid’s genome, replication integrates that “foreign” DNA into that genome: this process is the BASIS for molecular cloning!
- CRISPR-Cas9 uses the same technique that bacteria use to defend themselves against viruses to delete or induce mutations in genes. Inserting genes is especially tricky
- Experiments have shown that using the Gibson assembly process in addition to CRISPR-Cas9 allows for more precise insertions!
- Together, we can use these two things to do SO MUCH: potentially create biofuels, create biological systems to observe drug efficacy in the human body, and restoring and improving our pets!
Thanks for reading this article — if you liked it, make sure to give me a couple of claps! If you would like to connect with me, I’m on Linkedin and can be reached at my personal email: writetoapurva@gmail.com. Until next time!
