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,

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....

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 calculator

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 comb.......

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