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

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) toolkits^2,3 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 DNA^3,6. 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 plasmids^3,8. 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 bacteria^11,12,13, 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.

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 P_LuxB, 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.

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 P_LuxB, 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 calculator¹⁸, the second CDS is mTurquoise. Finally, the operon terminates with the terminator L3S2P21¹⁹.

Domestication of the Terminator
To assemble the designed polycistronic operons, all molecular parts need to be domesticated in the relevant acceptor plasmid. The terminator L3S2P21 was constructed for domestication by annealing two oligos (see Table S2). The oligos were designed with 4-nucleotide overhangs for insertion into pSL106. Figure 4A shows the result of a negative control, transforming a GG reaction with the acceptor plasmid pSL106 without an insert. Here, > 1000 red colonies and 10 white colonies were found. The red colonies likely have a mutation in the ccdB gene, whereas the white colonies are expected to be the result of self-ligation. Figure 4B shows the result of the actual promoter domestication. Here, 264 white colonies and no red colonies were found. From the plate shown in Figure 4B, two colonies were randomly picked for plasmid isolation. Through Sanger sequencing, they were confirmed to carry the correct insert.

Assembly of Level 1 plasmid
Level 1 of MoClo kits typically assembles multiple parts into transcription units. In the presented case of cloning a polycistronic operon, these Level 1 assemblies are incomplete transcription units, as one lacks a terminator and the other a promoter. At the next assembly Level, these partial transcription units form the polycistronic operon. A Level 1 plasmid was assembled from pSL069, annealed oligos JVO71 and JVO72, pSL842, and pJV092 (Figure 2). A negative control was included where only pSL069, annealed oligos JVO71 and JVO72, and pSL842 were included, thus missing a part for the terminator position. No transformants were obtained for this negative control reaction (Figure 5A). Figure 5B shows the result of the complete assembly reaction, yielding 292 white colonies and no red colonies. From the plate shown in Figure 5B, two colonies were picked for cultivation, miniprepped, and the insert was confirmed through Sanger sequencing.

Assembly of a multi-gene construct
The designed polycistronic operon was assembled from the two Level 1 plasmids pJV169 and pJV172, as well as the acceptor plasmid pMA60 and the end linker pMA668 (Figure 3). pJV169 and pJV172 release an intermediate assembly containing a CDS each, which assemble in a multi-gene construct in positions 1 and 2, respectively. Figure 6A shows a negative control where pMA60, the assembly for position 1 (pJV169) and pMA668 were included, but the assembly for position 2 (pJV172) was missing. The transformation of this reaction yielded 2 white colonies (incubation at 37 °C ). Figure 6B shows the results of the complete assembly of the multi-gene construct. 9 large and 435 small white colonies were present. The 9 large colonies are expected to be the result of self-ligation of the cut acceptor vector. The 435 small colonies are likely from correctly assembled plasmids. Their growth defect indicates that the construct imposes a burden. Figure 6C shows the same negative control as in Figure 6A, but incubated at 30 °C. Here, 4 white colonies were present. Figure 6D shows the assembly of the multi-gene construct, incubated at 30 °C, yielding > 1,500 colonies.

The assembly of the multigene-construct was confirmed through restriction digestion, induction of fluorescent protein expression, and nanopore sequencing. Figure 7 shows a BsaI test digest of plasmids extracted from 24 colonies randomly picked from the plate shown in Figure 6D. The BsaI digest of the acceptor plasmid pMA60 gives the expected single band at ~5,500 bp, as is seen in Figure 7. BsaI digest of the assembled multi-gene construct should give bands at ~4,300 bp and ~1,700 bp. This pattern was observed for 23 out of 24 colonies.

Correctly assembled constructs should convey OC-6 inducible GFP and mTurquoise expression. To check for inducible fluorescence, transformants of the operon assembly were replica plated onto a plate with and without the inducer. The fluorescence of both fluorescent proteins was imaged a plate imager capable of fluorescence imaging (Figure 8). Finally, the plasmid from colony 1 (see Figure 7) was Nanopore sequenced, confirming it carries the correctly assembled bicistronic operon.

Figure 1: In-Cloning Level 0: A schematic overview of the 12 Level 0 parts and their respective acceptor plasmids. The colored bars indicate the specific 3-nucleotide overhang that is created by the restriction enzyme SapI when a part is released. The Level 0 plasmids have a spectinomycin resistance marker, and the acceptor plasmids have a selection cassette containing a ccdB gene and an mCherry gene removed during cloning. For domestication in a Level 0 plasmid, the molecular part requires extensions that provide the recognition sites of BbsI (highlighted in gray) and generate overhangs (blue) for the appropriate acceptor plasmid by BbsI cleavage. The example shows the domestication of a terminator into pSL106. The generated overhangs are compatible with those of the BbsI-digested acceptor plasmid (pSL106), assembling a Level 0 terminator plasmid. The terminator can be released from its Level 0 plasmids by SapI, resulting in the overhangs TGA (light brown) and CGG (red). Please click here to view a larger version of this figure.

Figure 2: In-Cloning Level 1: A schematic overview of the 7 positions within a multigene construct, and the corresponding Level 1 plasmids. The used Level 1 acceptor plasmid determines in which of these 7 positions the insert will go during Level M/P assembly. Level 1 plasmids have an ampicillin resistance marker, and the acceptor plasmids have a selection cassette containing a ccdB gene and an mCherry gene removed during cloning. Each position has four corresponding acceptor plasmids that can release a transcription unit for either Level M or Level P in either forward or reverse orientation, allowing cloning in each orientation. The plasmids shown in this figure release a TU for further assembly in Level M or P. The acceptor plasmids can be digested by SapI, creating 3 nucleotide overhangs for assembly from Level 0 parts. The assembly in Level 1 plasmids, in turn, can be released by BbsI for further assembly in Level M. A cloning example is shown where a promoter + RBS, a CDS, and a bridge are released from their Level 0 plasmids and assembled into the acceptor plasmid pSL069 (position 2). The resulting assembly can be released by BbsI, creating overhangs for cloning into the second position (TU2) in a Level M/P acceptor. Please click here to view a larger version of this figure.

Figure 3: In-Cloning Level P/M: A schematic overview of the 7 positions of a transcription unit in a multigene construct and corresponding end linkers and Level M and Level P acceptor plasmids. The acceptor plasmid is chosen based on the first filled position (for each, there is a Level M and Level P variant), and the end linker based on the number of remaining positions to the right. Level P plasmids have a kanamycin resistance marker, and Level M plasmids have a spectinomycin resistance marker. The acceptor plasmids have a selection cassette containing a ccdB gene and a LacZα marker removed during cloning. All Level P and M acceptor plasmids have one overhang in common, GGGA, which is the 3' sequence of the end linker. The end linker also provides the Type IIS recognition site needed to release the assembled DNA construct for M/P cycling. Assembly from Level M into Level P uses BsaI, and from Level P into Level M uses BbsI. The cloning example shows the assembly of 2 TUs with positions 1 and 2. pMA60 is used as the acceptor plasmid, which starts the assembly at position 1. To complete the assembly of these two TUs into pMA60, the end linker LM-EL2 (pMA668) is used. Please click here to view a larger version of this figure.

Figure 4: Representative domestication of parts (Level 0 cloning). (A) A negative control with the acceptor plasmid pSL106 without an insert, resulting in ten white colonies and > 1000 red colonies. (B) Domestication of the L3S2P21 terminator into pSL106, resulting in 264 white colonies and no red colonies. Please click here to view a larger version of this figure.

Figure 5: Representative Level 1 assembly. (A) A negative control, omitting the terminator part, results in zero colonies. (B) Assembly of acceptor plasmid pSL069 with an RBS (JVO70+JVO71), mTurquoise (pSL842), and terminator (PJV092), resulting in 292 white colonies. Please click here to view a larger version of this figure.

Figure 6: Representative multi-gene assembly (Level M). As negative controls, the assembly reaction was performed, leaving out the Level 1 assembly destined for position 2 and transformed. (A) Negative control, incubated at 37 °C. (B) The complete assembly reaction (with the acceptor pMA60, Level 1 assembly for positions 1 (pJV169) and 2 (pJV172), and the end liner pMA668), transformed and incubated at 37 °C, gave 9 large and 435 small colonies. (C) Negative control, incubated at 30 °C. (D) Transformation of the complete assembly gave > 1500 colonies when incubated at 30 °C. Please click here to view a larger version of this figure.

Figure 7: BsaI restriction digest of 24 operon assembly clones. BsaI digest of the parental acceptor plasmid pMA60 gave a single band at ~5500 bp. For the correct assembly, two bands (~4300 bp and ~1600 bp) are expected. Of the assayed 24 colonies, 23 showed a pattern with two main bands of the expected sizes. Colony 13 only showed a single band at ~4300 bp. Please click here to view a larger version of this figure.

Figure 8: Fluorescence imaging of transformants of operon assembly. Of the plate shown in Figure 8D, two replica plates were made on LB agar with 100 µg/mL spectinomycin, with either no (Figure 8A and Figure 8C) or 10 nM N-(β-Ketocaproyl)-L-homoserine lactone (OC-6) (Figure 8B and Figure 8D). GFP fluorescence was detected in a plate imager by blue light excitation and a 527 ± 20 nm emission filter (Figure 8A-B), mTurquoise fluorescence by blue light excitation and a 510 ± 20 nm emission filter (Figure 8C-D). Please click here to view a larger version of this figure.

Acceptor Plasmid	GG Forward extension	GG Reverse extension
pSL099	NNNNNGAAGACNNTTTG	ATGTNNGTCTTCNNNNN
pSL100	NNNNNGAAGACNNTATG	GGGTNNGTCTTCNNNNN
pSL101	NNNNNGAAGACNNTATG	GGATNNGTCTTCNNNNN
pSL102	NNNNNGAAGACNNTATG	TGATNNGTCTTCNNNNN
pSL103	NNNNNGAAGACNNTGGG	GGATNNGTCTTCNNNNN
pSL104	NNNNNGAAGACNNTGGG	TGATNNGTCTTCNNNNN
pSL105	NNNNNGAAGACNNTGGA	TGATNNGTCTTCNNNNN
pSL106	NNNNNGAAGACNNTTGA	CGGTNNGTCTTCNNNNN
pSL252	NNNNNGAAGACNNTACT	ATGTNNGTCTTCNNNNN
pSL253	NNNNNGAAGACNNTTGA	TTGTNNGTCTTCNNNNN
pSL254	NNNNNGAAGACNNTTGA	TTGTNNGTCTTCNNNNN
pSL255	NNNNNGAAGACNNTTTG	CGGTNNGTCTTCNNNNN

Table 1: Extensions for domestication of Level 0 parts. There are 12 different Level 0 acceptors that can be used in In-Cloning (Figure 1). This table shows the extensions that need to be added to an insert for assembly in each Level 0 acceptor plasmid.

Supplementary Table 1: An overview of the oligos used in this protocol. Please click here to download this File.

Supplementary Table 2: An overview of the plasmids used in this protocol. Please click here to download this File.

Supplementary Figure 1: Representative overview of the cloning of a polycistronic operon through Level 0, 1 and M. A schematic overview of the domestication of 7 parts of the polycistronic operon is shown. 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. The 7 parts are assembled into 2 Level 1 constructs. The promoter, first RBS, first CDS, and the terminator bridge are assembled into pSL68, the second RBS, second CDS, and the terminator are assembled into pSL69. The 2 Level 1 constructs are assembled into a Level M construct into pMA60 with the end linker pMA668. Please click here to download this File.

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 others^3,6. The reusability of parts allows the creation of pooled or arrayed combinatorial libraries of multigene constructs. This facilitates, for instance, optimization of heterologous pathways with regard to finding ideal promoter strengths or enzyme identities. The standardized nature of the method makes it amenable to automation, allowing high-throughput assembly of DNA. Furthermore, the Out-Cloning scheme of the In- & Out-Cloning MoClo toolkit supports streamlined generation of acceptor plasmids with other origins of replication and selective markers, enabling researchers to assemble plasmids suitable for their organism of choice⁷.

Here the In- & Out-Cloning MoClo toolbox was used. In-Cloning allows for scarless assembly of transcription units and derived higher-order assemblies⁶. Initially, it was described as a structured method for the assembly of single-gene constructs, but not for polycistronic transcription units. However, polycistronic operons are frequently encountered in bacterial gene topology^11,12,13, and as such, there is a demand for their assembly with the same scalability and modularity as is available for single transcription units. This protocol has shown how a polycistronic operon can be cloned using the In- & Out-Cloning MoClo toolkit.

It should be noted that In-Cloning uses high copy number plasmids, which can lead to issues when cloning toxic genes, large constructs, or construct that pose a burden on cells, as seen in the assembled multi-gene construct in this protocol²⁰. An obvious solution is the use of plasmids with a low copy number, or incubation at a lower temperature, such as 30 °C^10,21. Furthermore, an essential step of the domestication of parts is the removal of recognition sites of type IIS enzymes used in the method. This means that certain sequences cannot occur, and one may have to introduce mutations, removing any used type IIS recognition sites, to use the MoClo method.

Overall, MoClo toolkits combines standardized assemblies and high versatility, making it suitable for many cloning purposes.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ampicillin	Merck Life Science	A9518
Bacterial agar	Fisher Scientific	P0011B
BbsI-HF	New England Biolabs	R3539
BsaI-HF V2	New England Biolabs	R3733
E. coli DH5α competent cells
Eppendorf Mastercycler Nexus Thermal Cyclers	Eppendorf	6333000014
EzDrop 1000 Micro-Volume Spectrophotometer	Blue-Ray Biotech	BRED-1000
GeneJET Plasmid Miniprep Kit	Thermo Scientific	K0503
In- & Out- Cloning Toolkit
Ligase buffer	New England Biolabs	B0216
NaCl	Fisher Scientific	BP358
New Brunswick Innova® 42/42R - Stackable Incubator Shaker	Eppendorf	M1335-0080
PhenoBooth+	Singer Instruments	PHB-007
SapI	New England Biolabs	R0569
Spectinomycin	Merck Life Science	S4014
Standard labware
T4 DNA ligase	New England Biolabs	M0202
Thermomix MM	B. Braun Biotech International
Tryptone	Fisher Scientific	LP0042B
Yeast extract	Fisher Scientific	LP0021B