Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Summary

This study describes methods for the T7-mediated co-expression of multiple genes from a single plasmid in Escherichia coli using the pMGX plasmid system.

Abstract

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.

Introduction

Co-expression of multiple proteins is increasingly essential, particularly in synthetic biology applications, where multiple functional modules must be expressed¹; in studying protein-protein complexes, where expression and function often require co-expression^2,3; and in characterizing and harnessing biosynthetic pathways, where each gene in the pathway must be expressed^4,5,6,7,8. A number of systems have been developed for c....

Protocol

1. Obtaining Genes of Interest

1. Design synthetic genes.
   1. Optimize a gene sequence for E. coli expression.
   2. Remove any problematic restriction sites from the sequence (AvrII, NdeI, EcoRI, and XbaI).
   3. Incorporate restriction sites for cloning; a 5′-NdeI site and a 3′-EcoRI site are recommended. Other sites can be used, if necessary; refer to the multicloning region of the selected plasmid (Figure S1-S4). If desired, include a 5′ or 3′ encoding tag for Western blot detection.
   4. Commercially order the designed gene.

Results

In this study, the goal was to co-express five proteins from a single plasmid. The five-codon optimized synthetic gene fragments encoding either N- or C-terminal hexahistidine tags were purchased commercially. The synthetic genes were amplified by PCR and individually cloned into a PCR-blunt vector and sequenced. To generate the polycistronic plasmid, the five genes of interest were first cloned into a suitable pMGX plasmid, pMGX-A. Figure 2 shows PCRBlunt-

Discussion

Co-expression of multiple genes is increasingly essential, particularly in characterizing and reconstituting complex, multigene metabolic pathways^3,4,5. The pMGX system makes multigene co-expression in E. coli routine^6,7,8 and accessible to diverse researchers. In this study, five proteins of interest were shown to be simultane.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Enzymes
Alkaline Phosphatase, Calf Intestinal (CIP)	New England Biolabs	M0290S
AvrII	New England Biolabs	R0174S
EcoRI	New England Biolabs	R0101S
NdeI	New England Biolabs	R0111S
XbaI	New England Biolabs	R0145S
Herculase II Fusion DNA Polymerase	Agilent Technologies	600677
T4 DNA Ligase	New England Biolabs	M0202S
Name	Company	Catalog Number	Comments
Reagents
1 kb DNA ladder	New England Biolabs	N3232L
4-20% Mini-PROTEAN TGX Stain-Free Protein Gels	Bio-Rad	456-8095
50x TAE	Fisher Thermo Scientific	BP1332-4
Agar	Fisher Thermo Scientific	BP1423-500
Agarose	Fisher Thermo Scientific	BP160-500
Ampicilin	Sigma-Alrich	A9518-5G
BL21 (DE3) chemically comeptent cells			Comeptent cell prepared in house
B-PER Bacterial Protein Extraction Reagent	Fisher Thermo Scientific	PI78243
dNTP mix	Agilent Technologies		Supplied with polymerase
Gel Extraction Kit	Omega	D2500-02	E.Z.N.A Gel Extraction, supplied by VWR Cat 3: CA101318-972
Glycine	Fisher Thermo Scientific	BP381-1
His Tag Antibody [HRP], mAb, Mouse	GenScript	A00612
Immobilon Western Chemiluminescent HRP Substrate	EMD Millipore	WBKLS0100
IPTG	Sigma-Alrich	15502-10G
LB	Fisher Thermo Scientific	BP1426-500
Methanol	Fisher Thermo Scientific	A411-20
Pasteurized instant skim milk powder	Local grocery store		No-name grocery store milk is adequate
Nitrocellulose membrane	Amersham Protran (GE Healthcare Life Sciences)	10600007	Membrane PT 0.45 µm 200 mm x 4 m, supplied by VWR Cat #: CA10061-086
Plasmid DNA Isolation Kit	Omega	D6943-02	E.Z.N.A Plasmid DNA MiniKit I, supplied by VWR Cat #: CA101318-898
pMGX	Boddy Lab		Request from the Boddy Lab Contact cboddy@uottawa.ca
Primers	Intergrated DNA Technologies		Design primers as needed for desired gene
Synthetic Gene	Life Technologies		Design and optimize as needed
Thick Blot Filter Paper	Bio-Rad	1703932
Tris base	BioShop	TRS001.1
Tween-20	Sigma-Alrich	P9416-50ML
XL1-Blue chemically competent cells			Comeptent cell prepared in house
Name	Company	Catalog Number	Comments
Equipment
BioSpectrometer	Eppendorf	RK-83600-07
Gel box - PAGE	Bio-Rad	1658005	Mini-PROTEIN Tetra Vertical Electrophoresis Cell
Gel Imager	Alpha Innotech		AlphaImager EC
Incubator-oven	Fisher Thermo Scientific	11-690-650D	Isotemp
Incubator-shaker	Fisher Thermo Scientific	SHKE6000-7	MaxQ 6000
Personna Razors	Fisher Thermo Scientific	S04615
Power Pack	Bio-Rad	S65533Q	FB300
Transilluminator	VWR International	M-10E,6W
Thermocylcer	Eppendorf	Z316091	Mastercycler Personal, supplied by Sigma
UV Face-Shield		18-999-4542
Waterbath	Fisher Thermo Scientific	15-460-2SQ
Western Transfer Apparatus	Bio-Rad	1703935	Mini-Trans Blot Cell