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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

Abstract

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

Introduction

Synthetic biology aims to engineer biological systems with new functionalities useful for pharmaceutical, agricultural, and chemical industries. Assembling large numbers of DNA fragments in a high-throughput manner is a foundational technology in synthetic biology. Such a complicated process can break down into multiple levels with decreasing complexity, a concept borrowed from basic engineering sciences1,2. In synthetic biology, DNA fragments usually assemble hierarchically based on functionality: (i) Part level: "parts" refers to DNA fragments with a specific function, such as a promoter, a coding se....

Protocol

NOTE: The hierarchical cloning protocol offered in this toolkit can be divided into three major steps: 1. Cloning part plasmids; 2. Cloning transcription units (TUs); 3. Cloning multi-gene plasmids (Figure 1). This protocol starts from the primer design and ends with applications of the cloned multi-gene plasmid.

1. Primer design for cloning the part plasmid (pYTK001):

  1. Design the forward and reverse primers containing flanking nucleotides TTT at the 5&.......

Representative Results

Here the results of four replicative multi-gene plasmids for β-carotene (yellow) and lycopene (red) production. One integrative multi-gene plasmid for disrupting the ADE2 locus was constructed, the colonies of which are red.

Cloning CDSs into the entry vector (pYTK001)
ERG20 was amplified from the yeast genome and the three carotenoid genes crtE, crtYB, crtI

Discussion

The MoClo based cloning kit developed by Lee et al. provides an excellent resource for quick assembly of one to five transcription units into a multi-gene plasmid either for replication or integration into the yeast genome. The use of this kit eliminates the time-consuming cloning bottleneck that frequently exists for expressing multiple genes in yeast.

We tested five different conditions for the digestion/ligation cycles of Golden Gate cloning with T4 DNA ligase. We found that 30 cycles of di.......

Acknowledgements

This work was funded by the Research Foundation for the State University of New York (Award #: 71272) and the IMPACT Award of University at Buffalo (Award #: 000077).

....

Materials

NameCompanyCatalog NumberComments
0.5 mm Glass beadsRPI research products9831For lysing yeast cells
Bacto AgarBD & Company214010Component of the yeast complete synthetic medium (CSM)
Bacto PeptoneBD& Company211677Component of the yeast extract peptone dextrose medium (YPD)
BsaI-HFv2New England BiolabsR3733Sa highly efficient version of BsaI restriction enzyme
CarbenicillinFisher Bioreagents4800-94-6Antibiotic for screening at the transcription unit level
ChloramphenicolFisher Bioreagents56-75-7Antibiotic for screening at the entry vector level
CSM-HisSunrise Sciences1006-010Amino acid supplement of the yeast complete synthetic medium (CSM)
DextroseFisher ChemicalD16-500Carbon source of the yeast complete synthetic medium (CSM)
Difco Yeast Nitrogen Base w/o Amino AcidsBD & Company291940Nitrogen source of the yeast complete synthetic medium (CSM)
dNTP mixPromegaU1515dNTPs for PCR
Esp3INew England BiolabsR0734Sa highly efficient isoschizomer of BsmBI
Frozen-EZ Yeast Trasformation II KitZymo ResearchT2001For yeast transformation
HexanesFisher ChemicalH302-1For carotenoid extraction from yeast cells
KanamycinFisher Bioreagents25389-94-0Antibiotic for screening at the multigene plasmid level
LB Agar, MillerFisher BioreagentsBP1425-2Lysogenic agar medium for E. coli culturing
LB Broth, MillerFisher BioreagentsBP1426-2Lysogenic liquid medium for E. coli culturing
LycopeneCayman chemicalsNC1142173For lycopene quantification
MoClo YTKAddgene1000000061Depositing Lab: John Deuber
Monarch Plasmid Miniprep KitNew England BiolabsT1010LFor plasmid purification from E.coli
Nanodrop SpectrophotometerThermo ScientificND2000cFor measuring accurate DNA concentrations
NotI-HFNew England BiolabsR3189SRestriction enzyme for integrative multigene plasmid linearization
Nourseothricin SulphateGoldbioN-500-100Antibiotic Selection marker for the pCAS plasmid used in this study
Phusion HF reaction Buffer (5X)New England BiolabsB0518SBuffer for PCR using Phusion polymerase
Phusion High Fidelity DNA PolymeraseNew England BiolabsM0530SHigh fidelity polymerase for all the PCR reactions
pLM494Addgene100539Plasmid used to amplify crtI, crtYB and crtE used in this study
Quartz CuvetteThermo Electron10050801For quantifing carotenoids
T4 ligaseNew England BiolabsM0202SLigase for Golden Gate cloning
ThermocyclerBIO-RAD1851148For performing all the PCR and cloning reactions
Tissue HomogenizerBullet BlenderModel: BBX24For homogenization of yeast cells
UV-Vis SpectrophotometerThermo ScientificGenesys 150For quantifing carotenoids
Yeast ExtractFisher BioreagentsBP1422-500Component of the yeast extract peptone dextrose medium (YPD)
β-caroteneAlfa AesarAAH6010603For β-carotene quantification

References

  1. Endy, D. Foundations for engineering biology. Nature. 438 (7067), 449-453 (2005).
  2. Engler, C., Gruetzner, R., Kandzia, R., Marillonnet, S. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enz....

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