How are DNA sequences assembled? (Artificial Gene Synthesis, Part 2)
Please welcome…molecular cloning!
In the process of genetic engineering where the goal is to give a target organism the genes of another, we need to first isolate and amplify the desired gene (via PCR), then put it into a binary vector that can then deliver the gene into the target organism’s tissues. Molecular cloning is how a gene (or multiple genes) is inserted into a binary vector (a circular ring of bacterial DNA modified for gene insertion). Because a solitary gene can’t be directly delivered into any plant or animal cell, this step facilitates the delivery of our desired gene into the target organism.
As we explore the topic of molecular cloning, I’ll be using some terms and concepts such as PCR, oligonucleotides, and primers, all of which I’ve defined in Part 1. So, if you haven’t already taken a look at that, I’d encourage you to do so to gain a better understanding of what we’ll be talking about today.
Molecular cloning is a broad term; it describes all the methods used to connect DNA segments together to build genetic constructs. There are four well-known molecular cloning techniques, but let’s focus on two of the most popular ones: Golden Gate Cloning and Gibson Assembly.
- Golden Gate Cloning
- Gibson Assembly
- Other molecular cloning techniques (Gateway, Yeast)
Golden Gate Cloning
Before we dive into the science here — yes, this method is named after the Golden Gate Bridge. This metaphor boasts its ability to seamlessly connect multiple DNA segments together, just like how the bridge connects two land masses together.
Let’s begin! In Golden Gate Cloning, DNA is first cut using enzymes at the spot where the foreign gene will be added, producing “sticky ends” (Fig 1). These sticky ends are single-stranded overhangs of DNA that protrude from the end of a double-stranded DNA sequence. If the sticky ends of two DNA segments have corresponding base pairs (A to T and C to G), they will naturally be attracted to each other and form hydrogen bonds between them. Basically, sticky ends stick.
But how exactly are sticky ends made?
Restriction Enzymes
An enzyme is a protein that is specialized to quicken metabolic processes. This broad category includes the star of Golden Gate Cloning: restriction enzymes, which exist to cut at one specific sequence of DNA like very precise and predictable scissors. Carrying a predefined DNA sequence, they search for a matching sequence along the DNA strand, and when they find it, snip!
A restriction enzyme can cut DNA in one of two ways that result in blunt or sticky ends. A restriction enzyme that makes blunt ends (like EcoRV) means it cuts straight through both strands of the DNA, while one that makes sticky ends (like EcoRI) creates an overhang of a few nucleotides on each side. The latter is the type of restriction enzyme that is needed for Golden Gate Cloning.
But it’s not usually as simple as using the same restriction enzyme to make sticky ends out of the two DNA segments you wish to connect. After all, two completely different genes are unlikely to share the same base pair sequence for you to slip a restriction enzyme into to cut and connect.
Instead, when trying to connect a gene isolated from PCR to a binary vector, the binary vector will be the one with the restriction enzyme-created sticky end while a corresponding overhang must be added to the solitary gene. Before that can happen, we must ensure that there is an overlapping section between the two segments. This can be done by editing primers, the short, single-stranded DNA segments we talked about in the previous blog. For example, if the sticky end on the binary vector is ‘TTAA’, we can make an overhang of ‘AATT’ on the primer, which will connect to the binary vector because they are complementary. Notice how the actual sequence of the gene to which this primer is attached is not ‘AATT’ — as you can see in Fig 3, it is ‘CAAT’. By making this change to the primer, we’ve effectively edited the end of this gene into a segment that will work as a sticky end!
Since PCR only produces double-stranded blunt ends, we won’t have a sticky end by the end of PCR; we’ll have a gene with an overlapping segment. To create the sticky end, we must use restriction enzymes to cut at this edited end with the same method discussed earlier.
Now, after the matching sticky ends of the binary vector and foreign gene bind together naturally, there is just one step left in Golden Gate Assembly.
As you can see in my beautiful drawing below (Fig 4), although the vertical base pairs of the two segments are connected, the highlighted areas between the horizontal lines (the sugar and phosphate backbone) are still detached. This is where the enzyme DNA ligase comes in to fill the gaps. Only then is the connection secure.
(And, in case you’re wondering, DNA ligase can be used to directly connect two blunt-end DNA segments; it’s just less efficient because no overhangs exist to align the two DNA molecules.)
And that’s a wrap! Golden Gate Cloning is a highly efficient and precise molecular cloning method that, because of its structure, can also be used to connect multiple genes to each other at once — as long as each sticky end corresponds to the following gene’s.
Next, let’s take a look at Gibson Assembly!
Gibson Assembly
This method operates with the same strategy of connecting corresponding sticky ends, but how those overhangs are made is completely different and there is an extra step in connecting the pieces together (ligation).
The image below (Fig 6) was what made it click for me as I was learning about this, so let’s walk through it together.
Step 1 — DNA design
In Gibson Assembly, the requirements for the DNA segments’ ends are a bit different. Here, we start with blunt ends for both the binary vector (purple) and foreign gene (teal). However, we still need overlapping gene sequences (yellow and red) at the ends so that the two segments can later align and connect at the right spots. These homology regions are made through PCR, where the primers for the foreign gene are extended to also match the first 15–40 base pairs of the binary vector at either side of the insertion point.
Step 2 — Exonucleases do their magic
I’ve introduced how, like Golden Gate Cloning, Gibson Assembly connects DNA sequences by attaching sticky ends together. But now we have a problem: how are we supposed to attach sticky ends together when the two DNA segments have blunt ends?
Well, in Gibson Assembly, sticky ends are made by 5' (“five prime”) exonucleases.
Let’s unpack this term: 5' means that it starts on the 5' side of a double-stranded DNA molecule, working towards the 3' end (Fig 8). “Exo” defines how it works outside or at the ends of a DNA molecule, as opposed to “endo,” which means it works from the middle of a DNA molecule (ex., restriction enzymes and CRISPR). “Nuclease” stands for “nucleotide” and means that it is a DNA degrading enzyme that “chews away” at a DNA strand, leaving disconnected single nucleotides in its wake. Altogether, “5' exonuclease” can be interpreted as “an enzyme that removes nucleotides from the 5' end of a double-stranded DNA molecule.”
Or maybe “remove” is too kind of a word — this enzyme furiously eats away at one strand on each side of the double-stranded DNA segments, expelling up to 80 nucleotides and thereby creating extremely long sticky ends.
Step 3 — Alignment
Of these excessively lengthy sticky ends, though, still only the 15–40 bp of homologous material defined by the primers match. As a result, these are the areas where the segments align — it ignores the other nucleotides eaten away by the exonuclease. So, we end up now with a connected yet very incomplete DNA molecule.
Step 4 — DNA Polymerases!
To fill in these gaps, let’s bring back DNA polymerases! They were the ones who gathered nucleotides and built all the new sequences during PCR. Here, they take on a very similar role. Attaching to the 3' ends facing the incomplete segments, they collect nucleotides floating around in the solution around them and string them together in the sequence laid out by the existing opposing strand. When they hit the point where the exonuclease stopped and the nucleotides are still attached, they disconnect, their work done.
Steps 5 & 6 — DNA Ligase, our favorite superglue, finishes the job
Just like how DNA ligase was needed in Golden Gate Cloning to connect the sugar/phosphate backbone of the DNA after the nucleotides were bonded, it is also needed now for the same reason.
After that, we have our desired gene seamlessly integrated into the binary vector! That’s a wrap for Gibson Assembly.
Yeast Assembly
Yes, yeast, the same little fungi that makes your bread rise and brews your beer. This inconspicuous organism that lives in your kitchen cabinet has hidden talents that make it a precious tool in biotechnology!
The creative molecular cloning technique of yeast assembly takes advantage of the natural abilities of yeast to repair its own DNA. Specifically, this system allows it to integrate foreign DNA into its genome smoothly and reliably. This method is called homologous recombination and requires the DNA fragments to share large overlapping areas of homology, which will recombine with each other and insert the desired gene. Basically, it works just like the method of Gibson Assembly, but inside yeast. So, unlike recombination in Gateway Cloning, there is no specific site or sequence that the fragments must have for recombination to occur — there just needs to be yeast!
Despite its using the same methods as Gibson Assembly, Yeast Assembly has a few significant differences. Well, everything’s just…bigger. Instead of having overhangs of up to 80 bp like what’s seen in Gibson Assembly, the exonucleases in Yeast Assembly create massive overhangs of about 1,000 bp long! As you can see, Yeast Assembly is better suited for the assembly of very large chunks of DNA. But this comes at a cost — in order for yeast to go through this process, an extra ~2k bp of components and genes must be added. In addition to the pain in the back this extra step is, a general rule in biotech is that the longer a piece of DNA is, the less efficient and reliable methods are in editing it. It’s because of these kinds of tradeoffs that so many different molecular cloning methods exist — each one hopes to provide the ideal solution for a specific situation, and as scientists, we must choose.
Other Molecular Cloning Methods
Restriction Enzyme Cloning, Gibson Assembly, and Yeast Assembly are all powerful tools for molecular cloning: they are both precise and efficient and can connect multiple genes at once, among other qualities that make them the top choices. However, a primary factor that can influence the decision between the two, for example, is the availability of binding sites for restriction enzymes since each type of restriction enzyme can only bind to one specific sequence of 4–8 bp. A gene sequence can lack the right binding sites, or have too many of them — if there is a binding site in the middle of a gene, a restriction enzyme would bind to that and cut the gene in half, something we definitely don’t want. (There are ways to remove problematic binding sites like these, but that could get complicated and laborious.) The number of restriction enzyme binding sites is just one of the many factors that can lead to a preference for alternative molecular cloning techniques. These four molecular cloning methods are some of the most popular, but there are dozens of other well-established techniques outside of that, and maybe hundreds more as this field continues to expand.
What’s next?
And here we have it: artificial gene synthesis! From PCR and primer design to the various methods of molecular cloning, we’ve covered it all. But, designing a binary vector that contains exciting new genes is only the first step in making transgenic plants. From here, we must use gene insertion methods to deliver the binary vector into the target cells and then cultivate those cells into full-grown plants.
It’s a long but exciting road, for who knows what will await you at the end? Bioluminescent flowers, purple tomatoes, the next tool to battle climate change? You name it!
