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Method Article

Standardized Modular Assembly of Polycistronic Operons with Modular Cloning (MoClo) using the In-Cloning toolkit

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DOI:

10.3791/68103

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September 2nd, 2025

 ,  ,  ,  , 
1Laboratory of Systems and Synthetic Biology (SSB), Wageningen University and Research, Agrotechnology and Food Sciences, 2Max Planck Institute for Terrestrial Microbiology, 3Center for Molecular Biology of Heidelberg University (ZMBH), 4Center for Synthetic Microbiology, Philipps-University Marburg

In This Article

Summary

This article presents a step-by-step protocol demonstrating how Modular Cloning (MoClo) can be adapted for the cloning of polycistronic operons.

Abstract

Modular Cloning (MoClo) toolkits enable the rapid assembly of multigene constructs. They are based on Golden Gate cloning, which uses Type IIS restriction enzymes that cut outside their recognition site. Since recognition and cut sequence are decoupled, overhangs created by Type IIS restriction enzymes can be deliberately chosen, and cloning strategies typically prevent cutting of correctly joined DNA fragments. This allows highly efficient assemblies of multiple DNA fragments in a single reaction. In MoClo, individual functional DNA parts such as promoters, ribosomal binding sites, coding sequences, and terminators, as well as higher-order assemblies, are assigned standardized overhangs, such that reusable parts libraries can be created. The majority of bacterial MoClo toolkits are designed for cloning monocistronic transcriptional units and do not provide a structured path for the assembly of polycistronic operons. This protocol demonstrates the assembly of polycistronic transcription units with the In- & Out-Cloning toolkit. The same approach is transferable to other MoClo toolkits.

Introduction

Molecular cloning, the introduction of recombinant DNA into a replicon such as a plasmid, is a foundational methodology in molecular biology. Construction and assembly methods of DNA have become increasingly sophisticated in recent decades. By now, researchers can build whole synthetic chromosomes and even genomes from synthesized DNA1. Traditional molecular cloning often relies on ad hoc cloning strategies, and can be laborious and expensive for large multi-gene constructs2. In recent years, Golden Gate (GG) cloning and derived hierarchical Modular Cloning (MoClo) toolkits2,3 have become popular solutions to allow time and cost-efficient DNA assembly.

Type IIS restriction enzymes are endonucleases that recognize a directional (non-palindromic) sequence and cleave outside this recognition sequence4. As a result, overhang sequences created by cleavage are not dependent on the recognition site, but can be chosen4. Further, functional DNA fragments, here called 'parts', can be designed such that cleavage removes the recognition site from the ends of to-be-joined parts2. GG cloning strategies leverage this to allow efficient multi-part DNA assemblies in one-pot reactions5. MoClo toolkits are based on GG cloning, and aim to provide a modular and hierarchical way for the assembly of multi-gene constructs. Overhangs used to assemble parts are standardized, allowing the creation of reusable and interchangeable parts libraries. MoClo toolkits rely on acceptor plasmids, which are engineered to accept multiple DNA sequences and release the assembled product. Each acceptor plasmid contains two recognition sites of two different Type IIS enzymes with one being used for accepting DNA cargo and the other for releasing assembled DNA3,6. An illustration of that principle is shown in Figure 1 for the cloning of an individual part, a terminator, into a MoClo acceptor plasmid (Level 0). Both the acceptor plasmid and the insert are cut by the restriction enzyme BbsI, in a way that leaves compatible overhangs and removes the BbsI recognition sites. Correctly ligated plasmids, thus, are not cut by BbsI anymore. Cutting the resulting plasmid with the type IIS enzyme SapI releases the previously inserted fragment in the next assembly step (Level 1).

In this protocol, the In- and Out-Cloning MoClo toolbox is used7. Here, Level 0 plasmids contain defined fundamental genetic parts such as promoters with 5' untranslated regions (UTRs), coding sequences, and terminators with 3'UTRs, Figure 1 shows a schematic overview of Level 0. On Level 1, multiple parts are being assembled into a transcription unit (TU), Figure 2 shows a schematic overview of Level 1 parts and cloning. In- and Out-Cloning MoClo has been designed for scarless assembly of transcription units in Level 1 by using a Type IIS enzyme that creates 3-nucleotide overhangs. This allows overhangs to be fully within the codon raster of the coding sequence, using the ATG start and the TGA stop codons for assembly. Levels M and P are used for the assembly of multiple transcription units released from Level 1 plasmids3,8. Figure 3 shows a schematic overview of the parts for cloning into Level M and P, and the assembly of a bicistronic operon in Level M (a bicistronic operon is a transcriptional unit that expresses multiple coding sequences (CDSs)). Level P and M have been designed to allow iterative cycling from one to the other, such that there is no theoretical limit on the number of parts that can be assembled, this however is not described in this protocol, as the main focus of this protocol is to show the assembly of a bicistronic operon. Although there is no limit to the number of parts that can be assembled, there is a limit to the size of a construct that can be assembled; up to 100,000 bp has been assembled using a MoClo kit9. Furthermore, large constructs or constructs that contain toxic genes may cause a burden on the cell; for these instances, modified protocols have been published10.

In- and Out-Cloning initially did not describe a scheme for the assembly of polycistronic transcription units. Polycistronic operons are a very common gene organization mode in bacteria11,12,13, and therefore, there is notable interest in their bottom-up assembly in a modular way14. Here, a structured scheme for the assembly of polycistronic operons using In- and Out-Cloning is described. The presented scheme allows facile swapping of the position of coding sequences within polycistronic transcription units, as well as assemblies consisting of both mono- and polycistronic transcription.

The following step-by-step protocol shows the design and cloning of a polycistronic operon using In- and Out-Cloning. The presented strategy for cloning polycistronic transcription units can easily be adapted to other MoClo toolkits.

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Protocol

NOTE: This protocol describes the design and assembly of a polycistronic operon through three Levels of the In-Cloning standard (0, 1, and M). The polycistronic operon assembled here starts with a promoter, followed by a ribosomal binding site (RBS), a coding sequence (CDS), a second RBS, a second CDS, and finally a terminator (Figure S1). It is strongly advised to use molecular cloning software to simulate the cloning in silico, to ensure the designed cloning strategy is correct. An example of assembling a promoter as a Level 0 part, assembling an RBS with a coding sequence and a terminator into a Level 1 Transcription Unit, and the assembly of a bicistronic operon in Level M is described here, and a schematic overview of the entire assembly is shown in Figure S1.

1. Level 0: Domestication of parts

NOTE: The term "domestication" is used to indicate that a molecular part has been integrated into a MoClo part library and can be assembled throughout the Levels of the MoClo kit. Typically, all recognition sites of Type IIS restriction enzymes used within the kit are removed from the to-be domesticated part, e.g., by synonymous recoding.

  1. Design of the insert
    1. Choose the molecular parts, such as promoter, CDS, and terminator, to be assembled together in the operon.
      NOTE: In this protocol the operon initiates with the promoter PLuxB, which can be activated by the protein LuxR in the presence of the inducer compound N-(β-Ketocaproyl)-L-homoserine lactone (OC6)17. GFP is the first CDS of the operon; its RBS is iGEM BBa_B0064. GFP is followed by a non-terminal linker, the 'terminator bridge'. The second CDS is mTurquoise. Its RBS has been designed by an RBS calculator18. Finally, the operon terminates with the terminator L3S2P2119.
    2. Choose the position in which the part should be assembled, an overview is shown in Figure 1. Based on the position, choose the acceptor plasmid to be used in the GG.
      NOTE: Parts can be domesticated for later use in various positions. For instance, a promoter can be assembled as a single part later on connecting with a 5'UTR part (pSL252), or it can be domesticated with a 5'UTR already contained (pSL099). A CDS can be assembled in four acceptor plasmids, to be assembled without a tag (pSL102), to be assembled with an N-terminal tag (pSL104), to be assembled with a C-terminal tag (pSL101), or to be assembled with both a N-terminal tag and a C-terminal tag (pSL103). In the given example, the promoter is domesticated into pSL252, the first RBS is domesticated into pSL253, the first CDS is domesticated into pSL102, the terminator bridge is domesticated into pSL106, the second RBS is domesticated into pSL099, the second CDS is domesticated into pSL102, and the terminator is domesticated into pSL106.
    3. Add extensions to the designed insert based on the chosen acceptor plasmid. Table 1 provides an overview of the extension required for GG assembly per acceptor plasmid. These extensions should be added during the design of the primers for PCR, the design of the oligos in case of annealing two oligos to construct a part, or during gene synthesis.
      NOTE: In order for a part to be domesticated, it is necessary to ensure that the part itself does not contain recognition sites of the Type IIS enzymes used within the kit, SapI (5'-GCTCTTC-3'), BbsI (5'-GAAGAC-3'), and BsaI (5'-GGTCTC-3'). The presence of recognition sites in parts drastically reduces the efficiency of correct assembly because the final product can be cut by the used Type IIS enzyme (see Figure 1).
  2. GG reaction
    1. For the GG reaction, add 50 fmol of the desired insert, 50 fmol of the acceptor plasmid, 1 µL of 10x T4 DNA ligase buffer, 1 µL of T4 ligase, and 1 µL of BbsI. Add nuclease-free water to a total volume of 10 µL. Additionally, include a negative control with the same reaction without the desired insert.
    2. Run the following program in a thermocycler: 25 cycles of 37 °C for 3 min (37 °C is the optimal temperature of the restriction enzyme) and 16 °C for 4 min (16 °C is the optimal temperature of T4 ligase), followed by a final digestion at 50 °C for 20 min (at 50 °C, T4 ligase shows very little to no activity, whilst the restriction enzyme is still able to function, thus limiting the amount of uncut acceptor plasmid in the final product) and a heat inactivation of the enzymes at 80 °C for 20 min (at 80 °C, all enzymes are heat inactivated and the reaction is terminated)6.
    3. Transform the GG reaction by adding 5 µL of the GG reaction in 50 µL of chemically competent E. coli cells and perform a heat shock transformation with a recovery step as described in a previously published protocol15.
    4. Spread 100 µL of the transformation outgrowth on a Luria-Bertani (LB) agar plate with 100 µg/mL spectinomycin. Incubate the plate overnight at 37 °C.
    5. Compare the transformation efficiency of the negative control to that of the complete assembly by counting the number of white colonies on both plates. Proceed to the next step if the assembly has about 10 times (or more) white colonies than the negative control.
      NOTE: The majority of resulting colonies should be white, with only a small fraction of red colonies. Acceptor plasmids in the In-Cloning toolkit have a dual selection cassette, containing either mCherry (Level 0 and Level 1) or LacZα (Level M and Level P), and a ccdB gene, encoding a gyrase inhibitor. Transformation of the GG reaction is performed in ccdB sensitive E. coli cells, thus, only cells that have the assembled plasmid can grow, whereas the original acceptor plasmid can not be propagated. Examples of ccdB sensitive strains include DH5α and TOP10. The mCherry/LacZα allows for visual identification of cells escaping the ccdB mechanism16.
    6. For each assembly, cultivate two white colonies by picking up some material of the colony with a pipette tip and resuspending it into 5 mL of LB medium with 100 µg/mL spectinomycin each and incubate overnight at 37 °C in a shaking incubator at 250 rpm. Perform a column-based plasmid extraction according to the manufacturer's instructions. Verify that the extracted plasmid has the expected sequence by Sanger sequencing.

2. Level 1: Determination of positions

NOTE: Level 1 is the Level between domesticating parts and assembling a polycistronic operon (Figure 2). Here, the positions of intermediate assemblies in the final construct are determined. The position of a Level 1 assembly within the operon can be altered by using a different Level 1 acceptor plasmid. The given example describes the assembly of a bicistronic unit, but the given scheme can be easily adapted for the assembly of up to six coding sequences (with an RBS each). Assembly of more coding sequences additionally requires cycling between Level P and M. With iterative cycling between Level P and M, there is no fixed upper limit to the number of coding sequences that can be assembled into a single operon.

  1. Choice of parts and acceptor plasmids
    1. Choose the level 0 parts to be assembled into a transcription unit. At a minimum, these need to include a promoter, a CDS, and a terminator.
    2. Choose the position in which the transcription unit should be assembled in the operon. Based on that position, choose the appropriate acceptor plasmid. An overview is shown in Figure 2.
      NOTE: The 7 DNA fragments for the construction of the bicistronic operon shown as an example are assembled into two Level 1 constructs. The promoter, first RBS, first CDS, and the terminator bridge are assembled into pSL68; the second RBS, the second CDS, and the terminator are assembled into pSL69.
  2. GG reaction
    1. For the GG reaction, add 50 fmol of each of the desired inserts, 50 fmol of the acceptor plasmid, 1 µL of 10x T4 DNA ligase buffer, 1 µL of T4 ligase, and 1 µL of SapI. Add nuclease-free water to a total volume of 10 µL.
    2. Run the following program in a thermocycler: 25 cycles of 37 °C for 3 min and 16 °C for 4 min, followed by a final digestion at 50 °C for 20 min, and heat inactivation of the enzymes at 80 °C for 20 min6.
    3. Optional: Add 0.5 µL of SapI to the GG reaction and incubate at 37 °C for 1 h.
      NOTE: SapI is not as stable as the other used type IIS restriction enzymes. It has been found that a digestion step with SapI after the GG reaction reduces the amount of colonies that carry the re-ligated acceptor plasmid. SapI has an isoschizomer, BspQI, which may not need this final digestion step.
    4. Transform 5 µL of the GG reaction in 50 µL of chemically competent E. coli cells and perform a heat shock transformation, preferably with a recovery step as performed in step 1.2.3.
    5. Plate 100 µL of the transformation outgrowth on an LB agar plate with 100 µg/mL ampicillin. Incubate the plate overnight at 37 °C.
    6. For each, cultivate two white colonies by picking up some material of the colony with a pipette tip and resuspending it in 5 mL of LB medium with 100 µg/mL ampicillin each. Incubate overnight at 37 °C in a shaking incubator at 250 rpm. Perform a column-based plasmid extraction according to the manufacturer's instructions.
    7. Confirm correct assembly through a restriction digest with BbsI, checking that the size of the cutout matches the expected size of the insert. Digest 1000 ng of plasmid DNA by adding 1 µL of the appropriate 10x digestion buffer and 1 µL of BbsI. Add nuclease-free water to a total volume of 10 µL. Incubate the reaction at 37 °C for 15 min.
    8. Perform gel electrophoresis by loading the digested plasmid on a 1% agarose gel, run the gel at 100 V for 35 min.
      NOTE: An in silico digestion of the expected plasmid can be performed to simulate an agarose gel to predict the result.

3. Level M: Assembly of a polycistronic operon

NOTE: In this step, the polycistronic operon is being assembled. In the presented case of a bicistronic operon, two Level 1 inserts are assembled in positions 1 and 2. Accordingly, the acceptor plasmid pMA60 is chosen. Further, an end linker is required. All Level M and P acceptor plasmids have the overhang GGGA as the second overhang. End linkers bridge between the insert and acceptor plasmid and enable assembly of a Level P construct into the Level M and vice versa. For a Level P assembly of two Level 1 inserts, the end linker pMA668 is used and will take the third and final position. An overview of Level M and P and a visualization of the cloning step are shown in Figure 3. With assembly solely on Level M (or P), operons of up to 6 coding sequences can be cloned (positions 1-6). Position 7 is being taken by the end linker. The assembly of a higher number of coding sequences in an operon is possible, but requires cycling to Level M (or P), which is not described in this protocol.

  1. For the GG reaction, add 50 fmol of each desired insert, 50 fmol of the acceptor plasmid, 1 µL of 10x T4 DNA ligase buffer, 1 µL of T4 ligase, and 1 µL of BbsI. Add nuclease-free water to a total volume of 10 µL.
  2. Run the following program in a thermocycler: 50 cycles of 37 °C for 3 min and 16 °C for 4 min, followed by a final digestion at 50 °C for 20 min and heat inactivation of the enzymes at 80 °C for 20 min6.
  3. Transform 5 µL of the GG reaction in 50 µL of chemically competent E. coli cells and perform a heat shock transformation with a recovery step as performed in step 1.2.3.
  4. Spread 100 µL of the transformation outgrowth on a LB agar plate with 100 µg/mL spectinomycin. Incubate the plate overnight at 37 °C, or at 30 °C if expecting a metabolic burden or toxicity of the assembly.
  5. For each assembly, make a culture from two white colonies. To this end, dip a pipette tip into the colony and use it to inoculate 5 mL of LB with 100 µg/mL spectinomycin. Incubate overnight at 37 °C or 30 °C in a shaking incubator at 250 rpm. Perform a column-based plasmid extraction according to the manufacturer's instructions.
  6. Confirm correct assembly through a restriction digest with BsaI, checking that the size of the cutout matches the expected size of the insert, as performed in step 2.2.7.
    NOTE: The construct may be too long to be covered by Sanger sequencing for confirmation. It can then be confirmed by whole-plasmid sequencing, which is available as a commercial service from several sequencing service providers.

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Results

Here, the results of a bottom-up assembly of a polycistronic operon from Level 0 to Level M are shown (Figure S1). The operon initiates with the promoter PLuxB, which can be activated by the protein LuxR in the presence of the inducer compound N-(β-Ketocaproyl)-L-homoserine lactone (OC6)17. GFP is the first CDS of the operon, its RBS is iGEM BBa_B0064. GFP is followed by a non-terminal linker, the 'terminator bridge'. The second RBS is designed by an RBS calculator

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Discussion

Molecular cloning can be time-consuming and cumbersome, especially with ad hoc cloning strategies. MoClo toolkits can improve molecular cloning by their modularity, hierarchical assembly and highly efficient one-pot reactions. They rely on DNA assembly through Golden Gate reactions. Thanks to standardized overhangs, libraries of molecular parts can be reused and shared with others3,6. The reusability of parts allows the creation of pooled or arrayed comb...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

TAL, STdV and DS were supported by the Max Planck Society within the framework of the MaxGENESYS project.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
AmpicillinMerck Life ScienceA9518
Bacterial agarFisher Scientific P0011B
BbsI-HFNew England BiolabsR3539
BsaI-HF V2New England BiolabsR3733
E. coli DH5α competent cells
Eppendorf Mastercycler Nexus Thermal CyclersEppendorf6333000014
EzDrop 1000 Micro-Volume SpectrophotometerBlue-Ray BiotechBRED-1000
GeneJET Plasmid Miniprep KitThermo ScientificK0503
In- & Out- Cloning Toolkit
Ligase bufferNew England BiolabsB0216
NaClFisher Scientific BP358
New Brunswick Innova® 42/42R - Stackable Incubator ShakerEppendorfM1335-0080
PhenoBooth+Singer InstrumentsPHB-007
SapINew England BiolabsR0569
SpectinomycinMerck Life ScienceS4014
Standard labware
T4 DNA ligaseNew England BiolabsM0202
Thermomix MMB. Braun Biotech International
TryptoneFisher Scientific LP0042B
Yeast extractFisher Scientific LP0021B

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Golden Gate CloningType IIS EnzymesDNA AssemblyMultigene ConstructsStandardized OverhangsRibosome Binding SiteHeat Shock TransformationAgarose Gel Electrophoresis
Standardized Modular Assembly of Polycistronic Operons with Modular Cloning (MoClo) using the In-Cloning toolkit
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