This post was written by Alfonso Timoneda, with significant contributions from Robert Hurt and Mohamed Soufi.
So you’ve learned about the Modular Cloning (MoClo) system and what it can do for you, and you’ve chosen one of the MoClo kits available through Addgene that suits your experimental purposes. But what now? MoClo really simplifies vector construction by combining restriction and ligation steps together in the same tube at the same time, however, it can prove itself to be quite the mental puzzle if you lack previous experience with it. Don’t worry, we’ve got you covered! Here you will find some useful tips to help you make your plasmid construction with MoClo a walk in the park.
Before you begin, be sure to check out Addgene’s MoClo guide for all the basics of the process. Also refer to Figure 1 for a visual overview!
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| Figure 1: General overview of the MoClo assembly process to produce a multigene construct. Figure exemplifies the extraction and use of a coding sequence of interestCDS Level 0 part from an Addgene MoClo kit, to construct a Level 1 vector with a promoter and terminator flanking the CDS, and a Level 2 multigene vector with two other independently regulated transcription units. Created with BioRender.com. |
Planning makes perfect
Our first tip, and arguably the most important, is to take some time to sit down, plan your cloning, and have a clear vision of the final vector structure you want to achieve before starting your experiments. Don’t dive directly into the pipetting! MoClo nomenclature and structure can be quite confusing for new users, and your cloning success depends directly on the choices you make during the early stages of the process. Some questions to start with are:
- Is your sequence of interest a promoter, a coding sequence, a terminator, or other plasmid part?
- Do you want to fuse your protein to any tags or fluorescent markers?
- If you’re creating a multigene construct, in what position do you want your gene to be in relation to the rest of the genes?
- And do you want it to be in the same (forward) or in the opposite (reverse) orientation?
The answers to questions like these will determine which acceptor vectors and parts you will be using during the different levels of your MoClo assembly.
Know your process
The MoClo system allows for directional assembly of DNA sequences, called modules or parts. Assembly is dictated by four-base overhangs that are paired with different part types (e.g. 5’-GGAG and 3’-TACT flanking promoter parts, 5’-AATG and 3’-GCTT flanking CDS parts, etc). These overhangs are known as the common MoClo nomenclature. The overhangs are generated using type IIS restriction enzymes (BsaI, BbsI/BpiI, BsmBI), which recognize asymmetric DNA sequences and cleave at a short distance from their recognition sequence. While developing the MoClo system, researchers wisely agreed on universally using the same four-base overhangs for the same type of parts to ensure that these would be interchangeable between different laboratories. Therefore, choosing acceptor vectors and parts with the adequate overhangs is crucial for the position and directionality of the sequence you are trying to clone.
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| Figure 2: MoClo assemblies depend on compatibility of four-base overhangs flanking each genetic part. (A) Example Level 0 assembly. BpiI recognition sites and four-base overhangs flank a coding sequence of interest (CDS1, top). Overhangs must match the Level 0 acceptor (bottom) for CDS1 parts. (B) Example Level 1 assembly. The choice between different Level 0 vectors depends on the four-base overhangs flanking each part, as they need to match sequentially in order to properly ligate. PRO, promoter; CDS1, coding sequence with stop codon; CDSns, coding sequence with no stop codon; TAG, C-terminal tag; TER, terminator. Created with BioRender.com. |
The Sainsbury Laboratory Golden Gate Cloning videos made by the Nicola Patron group, one of the developers of the MoClo system, are an invaluable resource for learning about MoClo in an in-depth and easy-to-understand guided manner. These videos can be especially useful when designing new primers to clone your sequence of interest into Level 0 parts.
Use in silico cloning tools
When available, always perform your MoClo assemblies in silico before you start doing your cloning. Most sequence analysis software includes Golden Gate cloning tools that simulate the restriction–ligation reaction. You can input the sequences of the parts and acceptor vectors, as well as the restriction enzymes, and it will create the resulting plasmid map and identify any mismatches that are preventing your overhangs from aligning properly. Do this sequentially for your Level 0, Level 1, and Level 2 assemblies using the plasmid sequence files provided with every Addgene MoClo kit through their online catalog pages. We guarantee this will save you a lot of headaches down the line!
Cash in on gene synthesis
Before you start your cloning, it is advisable to remove unwanted type IIS restriction sites within your sequence to improve assembly efficiency. This can be done through domestication, which is the modification of a DNA sequence to remove a restriction enzyme recognition site without changing the function of the sequence, normally through the synonymous mutation of a codon. You can domesticate the sites yourself using PCR, but gene synthesis can save you time. Using gene synthesis, you can request the sites to be domesticated through the process, and request your sequence with the appropriate flanking restriction sites and overhangs already added. This way your synthesized DNA fragment can be used directly in your assemblies.
Preparing reagents
Keep it cool
You’ve done all the learning and planning, and your MoClo kit has just arrived! MoClo kits from Addgene will most likely arrive in your laboratory as plates of frozen glycerol stocks. It is very important that these stay frozen at -80 °C, since multiple rounds of freezing and thawing will affect bacterial stock viability. When handling your MoClo plate, always do so in cold conditions and preferably on dry ice.
Careful use of your glycerol stocks
When your lab adopts the MoClo technology, you will quickly go through your plasmid stocks. To avoid cross-contamination, we recommend retrieving plasmids from your original Addgene kit plate as few times as possible and always under sterile conditions. It’s best to grow new bacterial cultures from your kit plate and create your own glycerol stocks for more frequent use. This way, if one of your new glycerol stocks gets contaminated, you can resort back to your original Addgene kit plate for fresh bacteria.
Performing your Assemblies
Calculate your volumes
As you progress through the MoClo assembly levels, you will be using an increasing number of parts in your one-pot one-step reactions and your ligation efficiency will decrease. Furthermore, the size of the parts also impacts ligation efficiency, as concentration does not equal the number of DNA molecules. A solution of a longer DNA fragment will contain fewer molecules than a solution of a shorter DNA fragment with the same concentration. What really matters is the molar ratio of your fragments. To increase your ligation efficiency, we recommend you calculate the right volume of each plasmid according to their concentration, size, and the desired insert-to-vector ratio using the equation in Figure 3.
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| Figure 3: Calculation for the plasmid volume for MoClo experiments. The term ‘insert’ refers to plasmids carrying each part, ‘vector’ refers to acceptor plasmids, and the ‘amount of vector’ and the ‘molar ratio of insert to vector’ can be defined by you. |
Save time, go high throughput
You are ready to perform your assemblies, but you have a high number to get through. It’s time to think big and perform your assemblies in a high-throughput format. If your lab owns a liquid handling robot, you can set it up to automate the reagent transfer and the restriction–ligation reactions in a 96-well (or higher!) format. Liquid handling robots are not only faster and more accurate, but they also work with very small volumes of reagents which lowers experimental costs.
If you are planning to perform a high number of assemblies, you will also likely need to miniprep a lot of plasmids from your bacterial stocks beforehand. You can also use high-throughput methods to streamline your plasmid extractions. Use this protocol shared by Dr. Rob Hurt for high-throughput minipreps in 96-well plates.
Plate wisely
You will likely see fewer numbers of colonies (and therefore positive colonies) after each consecutive assembly and transformation round, due to the decrease in ligation efficiency. To combat this, you can increase the volume of competent cells you plate after each transformation and/or use different E. coli strains with higher transformation efficiency. If you’re cloning in high-throughput, we recommend transforming into chemically competent cells in a 96-well PCR plate.
Always confirm your results
Congratulations! You have followed these tips, and you now have what look like positive colonies on your transformation plates. However, even though the MoClo system is generally very efficient and precise, positive colonies may contain plasmids with undesired ligations, so always screen multiple colonies after each transformation. This could be as few as 1–2 colonies if you’re an experienced MoClo user, but beginners or those performing complex assemblies should screen a minimum of 8–10 colonies. You can check colonies either by PCR, by directly extracting and sequencing the plasmid, or both! We strongly encourage you to sequence the entire insert region of your final plasmid or those you are selecting for further assemblies. This will help confirm each part has ligated in the right order and direction (which is not easy to see in colony PCR electrophoresis), is free of undesired mutations, and that you have the correct four-base overhangs for future assemblies in the right locations.
We hope these tips will help you in your MoClo quest, so good luck and happy cloning!
Resources
Additional resources on the Addgene blog
- Plasmids 101: Modular Cloning
- Plasmids 101: Modular Cloning Applications and Kits
- Plasmids 101: Golden Gate Cloning
Additional resources on addgene.org
Topics: Plasmid Technology, Plasmids 101, Plasmid Cloning
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