NEB Podcast #55 -
Transcript
Interviewers: Lydia Morrison, Marketing Communications Writer & Podcast Host, New England Biolabs, Inc.
Interviewee: Andrew Sikkema, Research Scientist I, New England Biolabs, Inc.
Lydia Morrison:
Welcome to the Lessons from Lab and Life podcast, brought to you by New England Biolabs. I'm your host, Lydia Morrison, and I hope this podcast offers you some new perspective. Today, I'm joined by Andy Sikkema, who is a research scientist in the Lohman Lab here at NEB. The Lohman Lab studies the biological function of enzymatic mechanisms of DNA ligases and nucleases, which are the enzymes that cut and paste DNA. Andy is here to talk with us about an application of cutting and pasting DNA, and that is the DNA assembly method called Golden Gate Assembly, which can be used to join many fragments of DNA in a specified order in a single reaction. Andy, thanks so much for taking time out of your schedule to join us here in the podcast studio today.
Andy Sikkema:
Great. Thanks for having me.
Lydia Morrison:
Yeah, I'm so excited you can be here because we're in the middle of our Molecular Cloning 101 series for those individuals who might be new to molecular cloning, and you are here today to talk to us about Golden Gate Assembly. So could you just tell us what Golden Gate Assembly is to start?
Andy Sikkema:
Yeah, so Golden Gate Assembly is a restriction enzyme-based cloning method where you can assemble multiple DNA fragments in a predetermined order in a single reaction. So this is in contrast to the normal Type IIP restriction enzymes that are used in traditional cloning, which have a palindromic recognition site. Golden Gate utilizes Type IIS enzymes, which have a separate recognition and cleavage site, and that allows you to generate overhangs of any sequence. So if you carefully select the overhang sequences that you generate, you can assemble even dozens of fragments into single reactions in a specified order.
Lydia Morrison:
That sounds really efficient. Why would someone need to use Golden Gate Assembly?
Andy Sikkema:
So Golden Gate's really useful for designing molecular systems and is, really, frequently used in synthetic biology applications where the goal is to mix up things like promoters, genes, tags, terminators, and so by having those defined overhangs, which we usually call fusion sites, defined between all those different elements, you can mix and match all those parts. So you can essentially have all of your promoters have the same overhangs on each end, and that allows you to just pick whichever promoter you want out of your pool, and you can put them all together and do these big combinatorial screens of different parts. It's also really good for single-insert cloning because you don't have to go through the traditional thing of predigesting and then purifying your parts. You can just throw everything into one tube and do one reaction, and because of that, there's a cut step at the end of the assembly process that really knocks down the background. So you tend to get very low empty vector backgrounds in Golden Gate reactions as well.
Lydia Morrison:
So why would someone choose Golden Gate Assembly over another molecular cloning method?
Andy Sikkema:
So in terms of applications, there can be a lot of overlap between Golden Gate and other multi-insert methods like NEBuilder or Gibson Assembly®, but there are some important distinctions. Potentially the biggest is that Golden Gate has the ability to assemble many more parts in a single reaction, so our work has shown that you can pretty reliably assemble up to about 30 parts with a single Golden Gate reaction. The practical limit for something like Builder or Gibson is less than 10, so you have a lot more complexity you can do with that number of parts. The other big advantage to Golden Gate is that since it's using restriction enzymes to generate, generally, four nucleotide overhangs, it's really good at tolerating repetitive and structured sequences. That can be really challenging with something like NEBBuilder, which generates about a 20-nucleotide single-stranded overhang, and so that can potentially fold up. You can get all kinds of weird things happening with structured and repetitive sequences there. Because the overhangs are so short, with Golden Gate, you can usually get around that.
Lydia Morrison:
Okay. Those sound like some real advantages. Are there different ways of performing Golden Gate Assembly or are there different sort of variations to the method?
Andy Sikkema:
Yeah, so Golden Gate can be used for everything from single-insert cloning to multi-fragment combinatorial libraries and anything in between. A really common way that it's done is to do these standardized hierarchical assembly methods, referred to as MoClo-type cloning, where you organize assemblies in multiple rounds. So in the first round, you assemble individual transcriptional units, so that's like a promoter, a gene, a terminator, tags, et cetera. Then in the second round, you put multiple transcriptional units together into pathways, and then you can even do a third round to make even bigger constructs. With combining 3 to 5 parts per round, you can actually get very large constructs by the time you do three rounds of assembly.
Lydia Morrison:
Wow, that's pretty amazing. You mentioned combinatorial assembly. What can you tell me about that? Why would somebody do that, and do we have any recommendations for someone who's doing that?
Andy Sikkema:
Yeah. So Golden Gate is a fantastic method if you're interested in doing combinatorial assembly because it is very modular and has a really high fragment capacity, and you can do these things all in essentially one pot. Really, the main challenge with doing a combinatorial assembly with Golden Gate is actually finding a way to screen the library after you create it. So say you have a 10-part assembly with 10 variant of each part. That's actually a theoretical complexity of 10 to the 10th or 10 billion different combinations, and so you probably made the thing you want in there. The trick is finding it. So from the assembly side, these things are actually fairly straightforward.
You essentially just design your framework assembly to make sure all your junctions are fixed, and then you just come up with all the variance of the parts that you want to put into it, and then you work out the ratios that you want, put it all in a pot, assemble it, and then you can transform it and then try to find out how you're screening it. That's really the challenge. So there's a lot of these large gene foundries that use these methods, and most of their technology is not necessarily in the assembly. It's actually in the screening. I would actually say, if you want to do something like this, I would work backwards from come up with a good screen first and then figure out what you want to do for the combinatorial assembly after.
Lydia Morrison:
Great advice. Are there other tools or tips that would make Golden Gate Assembly easier for someone who was attempting it for the first time?
Andy Sikkema:
Yeah, so I'll cover that in a couple of different aspects. One of them, and the main limitation that comes up with Golden Gate, is that you have to domesticate the sequences. Domestication is the term we use for removing the native Type IIS sites from the sequence. So just like any restriction enzyme, you're going to see occurrences of the recognition motif in the sequences that you're working with, and those have to be removed so that they can be used. Most of the enzymes that are used for Golden Gate have 6-base pair recognition sites, but we just released a new enzyme called PaqCI that has a 7-base recognition site, so that means that we should see approximately one quarter the number of recognition sites for PaqCI one for most enzymes, and so if you're in positions where you don't want to deal with domestication because of your DNA sources, just having that ability to have fewer sites around is really helpful.
The other things that can be really helpful with this is NEBridge® tools that we've released, so building on our research work that allowed us to push the complexity of Golden Gate and do things like assemble the T7 genome in a single pot, we created a bunch of tools that we've now released, and so they can be found in two places. There's the NEBridge Golden Gate tool, which is a visualization tool that you can use to check your assembly products, look for internal sites. It'll even suggest primers for you. There's also the Ligase Fidelity Tools, which is great for diagnosing problems, so you can check the fidelity of your overhang to look for, maybe, incorrect ligations that can be holding your assemblies back. It's also really good for designing new assembly standards or modifying existing standards.
Then the last thing that has come out recently from NEB is the NEBridge Ligase Master Mix. So this is a kit that was developed that allows you to use all the common Type IIS enzymes with a single enzyme mix, so that contains the ligase and all the reaction components you need for the assembly. You just add the Type IIS enzyme you want to use, and you're off. So it really simplifies it. It cuts down on the number of components that you have to keep on hand.
Lydia Morrison:
Awesome. Thank you for sharing those great resources, the tools, and the products with our listeners. We'll make sure we put links in the transcript for this podcast to all of those resources so that our listeners can find them easily. I was curious if you have an interesting story that you could share about one of our customer's experiences related to Golden Gate.
Andy Sikkema:
So a lot of this technology is still relatively new, so things are just really starting to develop. The most probably high-profile thing that's come out using a lot of the tools that we've put out there is this paper called FRAGLER from, I believe, AstraZeneca®, and they use the tools that we built to build this platform that allows them to do these combinatorial assemblies internally. I think this is the first, but I have no doubt that this won't be the last. I think we're going to see a lot more of these kinds of things built on top of some of this ligase fidelity work coming out in the future.
Lydia Morrison:
Yeah, that's great. I can't wait to see what our collaborators come out with next. Okay, Andy, before I let you go, what are your top three tips for someone new to performing NEBridge Golden Gate Assembly?
Andy Sikkema:
So the three most important things somebody can do is check your assembly design, double check your assembly design, and then triple check your assembly design before you even start doing anything at the bench. Spending that in silico time making sure that your assembly is exactly what you want, you're making the parts that you think you want before you order primers and do a PCR is going to save you a lot of time because even internally, we have cases where we think we have everything ironed out. The assembly fails, and then we have to go back through and try to figure out what happened. It's usually just, we missed a nucleotide somewhere. We made a mistake in some part of the assembly. So spending that extra couple hours to go through and just really make sure that you have exactly what you want will save you a lot of headache and a lot of time later on.
The other issues that come up a lot is missing internal recognition sites. So if you fail to properly domesticate the sequences that you're working with, your products are all going to get cleaved at the end of the reaction, and you're really not going to get anything. Another common one is not checking the fidelity of the assembly. So we see this a lot going through, and just using NEBridge tools online and making sure that the assembly overhangs that you're using are giving you what you want can make a huge difference. We see a lot of tech cases come through where somebody has just missed something, like they put a palindromic overhang in their sequence, and that'll just completely kill your fidelity of the assembly.
Other things you can do is use good protocols. NEB has spent a lot of time, a lot of our development scientists have put a lot of time and quite a few years into developing the enzymes, the buffers, and the protocols used in Golden Gate assemblies, and they give really good results, so follow their recommendations. Finally, DNA quality, this is one that I think gets overlooked a lot. Making sure that you do your PCRs, you make sure that they're as good as you can possibly get them, and then clean them up properly before quantifying them and then using them in their reaction can make a big difference. We have some data where just having one contaminated part in an assembly can completely ruin the assembly efficiency and drop it by a thousand fold.
Lydia Morrison:
Wow, that's a big drop. Okay, folks, you heard it here first. Check your assembly design before proceeding. Super important. Also, don't be afraid to reach out to NEB's knowledgeable technical support staff if you have any questions. If you are new to Golden Gate Assembly and you need a hand, you need guidance beyond the online tools we offer and the online protocols we offer, you can always reach out to NEB technical support at info@neb.com, or you can find our technical support form on our website, www.neb.com. Thank you so much for joining me today, Andy. I know I've learned a lot. I'm sure our listeners will learn a lot, and I hope everybody goes and checks out the helpful resources that Andy's helped provide. I wish you success in your future at Golden Gate assemblies.
Andy Sikkema:
Thank you. It was great talking to you, and good luck on your assemblies too, listeners.
Lydia Morrison:
Thanks for joining us for this episode of the Lessons from Lab and Life podcast. I'd like to take a moment to remind you where to find some of the helpful resources we mentioned in this conversation. Head over to our website, neb.com, to find video tutorials and webinars as well as the answers to some questions our tech support team is frequently asked.
