MoClo Baculo toolkit

Developers: Zhihao Lai and Marion Boudes.

 

Introduction

 

The modular cloning (MoClo) Baculo toolkit is designed for constructing multigene expression vectors for the baculovirus system and, through compatibility with the MoClo Yeast toolkit, also for yeast. Vector construction by MoClo Baculo utilises Golden Gate assembly, which does not require PCR, primers or the sequencing of intermediate products. For users of the baculovirus system, MoClo Baculo is compatible with the Bac-to-Bac and biGBac systems.

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Protocol:

 

A step-by-step protocol can be found here. While the protocol does not mean to replace any of the papers under ‘recommended reading’ below, it does include all the required information to build multi-gene baculovirus expression vectors using MoClo Baculo. Since we assumed that most users of MoClo Baculo will be interested only in baculovirus expression, we included in the protocol only information related to the baculovirus system. Users who are interested in building yeast expression vectors are invited to read Lai et al. 2025 and Lee et al. 2015 (see references under ‘recommended reading’ below).

 

MoClo Baculo paper:

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MoClo Baculo toolkit:

Zhihao Lai, Sarena F Flanigan, Marion Boudes, Chen Davidovich. Modular Cloning of Multigene Vectors for the Baculovirus System and Yeast. J Mol Biol. 2025 Apr 1;437(7):168943. doi: 10.1016/j.jmb.2025.168943.

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Further recommended reading:

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MoClo Yeast toolkit (AddGene Kit #1000000061): 

Michael E Lee, William C DeLoache, Bernardo Cervantes, John E Dueber. A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth Biol. 2015 Sep 18;4(9):975-86. doi: 10.1021/sb500366v.

 

Modular cloning:

Ernst Weber, Carola Engler, Ramona Gruetzner, Stefan Werner, Sylvestre Marillonnet. A modular cloning system for standardized assembly of multigene constructs. PLoS One. 2011 Feb 18;6(2):e16765. doi: 10.1371/journal.pone.0016765.

 

biGBac system (AddGene Kit #1000000088):

Florian Weissmann, Georg Petzold, Ryan VanderLinden, Pim J Huis In 't Veld, Nicholas G Brown, Fabienne Lampert, Stefan Westermann, Holger Stark, Brenda A Schulman, Jan-Michael Peters. biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes. Proc Natl Acad Sci U S A. 2016 May 10;113(19):E2564-9. doi: 10.1073/pnas.1604935113.

 

Q&A:

 

Does the MoClo Baculo toolkit include all the plasmids I need?

Yes, if the intended application is only baculovirus expression, then we recommend that users get only the MoClo Baculo toolkit. The MoClo Baculo toolkit includes all the plasmids required to construct multi-gene baculovirus expression vectors compatible with the Bac-To-Bac system. Users who are interested in expressing 7 proteins or more from the same vectors will also need a couple of plasmids from the biGBac system (AddGene Kit #1000000088; see protocol for details). Of course, you will have to generate your open reading frames and possibly tags that you wish to use, but the protocol includes instructions for that.

 

Should I use the MoClo Baculo toolkit if I am only interested in baculovirus expression, not yeast?

Yes. We designed the MoClo Baculo toolkit under the view that most users would be interested only in baculovirus expression. We consider MoClo Baculo simpler to use and more robust than other approaches for vector building, aiming for expression using the baculovirus expression.

 

Is MoClo Baculo easy to use?

Yes and no. While Modular Cloning (MoClo) is easier and more robust than most other cloning approaches, it is sometimes harder to understand and plan for the first time. This is because MoClo includes more components and steps than other vector building methods. We recommended dedicating a couple of days to reading and understanding the concept of Modular Cloning and its implementation in the MoClo Baculo toolkit before starting a new project. Once the method is understood, we find the bench work commonly easier, quicker, more robust and requires less troubleshooting and sequencing than other cloning approaches.

 

My lab already uses the Bac-to-Bac system for baculovirus expression. Can I use MoClo Baculo-constructed vectors to express proteins the way I normally do?

Yes. MoClo Baculo is compatible with the Bac-to-Bac system. Therefore, all the protein expression vectors (Level 1, 2 and 3) include a backbone that is functionally equivalent to the pFastBac1 vector and are fully compatible with the Bac-to-Bac system.

 

I routinely use the biGBac system and love it. Is there any reason for me to try MoClo Baculo?

Yes. MoClo Baculo is compatible with biGBac, so you can benefit from the advantages of both systems: MoClo Baculo will allow you to construct your entry vectors quickly without PCR or primers, including quickly swapping tags in and out as needed. You could then combine multiple MoClo Baculo-constructed vectors into large biGBac plasmids using Gibson Assembly, as you are used to, given the compatibility between the systems (see protocol for details).

 

Can I use my lab’s repertoire of open reading frames for the MoClo Baculo toolkit?

Open reading frames can be used to build baculovirus expression vectors only if they are devoid of BsaI, BsmBI and Pmel sites. This is because the MoClo Baculo system utilises BsaI and BsmBI for Golden Gate assembly during vector building. Pmel is used for compatibility with the biGBac system. If you also intend to use the MoClo Baculo toolkit to build yeast expression vectors, then you will also need to remove NotI sites. 

 

I intend to use the MoClo Baculo toolkit for protein expression using both baculovirus and yeast. Do I need to get the MoClo Yeast toolkit?

Yes. Users who wish to benefit from the compatibility between the MoClo Baculo toolkit and the MoClo Yeast toolkit, aiming for both baculovirus and yeast expression, need to get both the MoClo Baculo toolkit and the MoClo Yeast toolkit (AddGene Kit #1000000061).

 

Are there any special reagents that I will need for using the MoClo Baculo toolkit?

MoClo Baculo is based on Golden Gate assembly, and also Gibson Assembly for extremely large vectors that express 7-30 proteins and/or are larger than 20-30 kbp. Hence, you will need reagents for Golden Gate assembly and Gibson Assembly, all of which are commercially available and specified in the protocol.