Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

Summary

The study describes a protocol for creating large (µg-mg) quantities of DNA for protein screening campaigns from synthetic gene fragments without cloning or using living cells. The minimal template is enzymatically digested and circularized and then amplified using isothermal rolling circle amplification. Cell-free expression reactions could be performed with the unpurified product.

Abstract

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins in less than 24 h using cell-free expression. After receiving DNA from a vendor, the gene fragment is PCR-amplified, cut, circularized, and cryo-banked. A small amount of the banked DNA is then diluted and amplified significantly (up to 10⁶x) using isothermal rolling circle amplification (RCA). RCA can yield microgram quantities of the minimal expression template from picogram levels of starting material (mg levels if all starting synthetic fragment is used). In this work, a starting amount of 20 pg resulted in 4 µg of the final product. The resulting RCA product (concatemer of the minimal template) can be added directly to a cell-free reaction with no purification steps. Due to this method being entirely PCR-based, it may enable future high-throughput screening efforts when coupled with automated liquid handling systems.

Introduction

Cell-free gene expression (CFE) has emerged as a powerful tool with many applications. Such applications include disease detection^1,2,3,4,5,6, micronutrient and small molecule detection^{7,8,9,10,11,12}, biomanufacturing^13,14

Protocol

1. Designing the gene fragment

NOTE: The gene fragment should have all the necessary genetic elements for transcription/translation, including promoter, ribosome binding site (RBS), start codon, the gene of interest, and terminator. While the terminator is not necessary for a linear expression template (LET), it will be important if the user decides to insert the sequence into a plasmid. These sequences were lifted from the pJL1-sfGFP plasmid⁵⁵ (gift from Michael Jewett&#.......

Discussion

The gene of interest can be any desired protein, but it is best to start with a fluorescent protein as a convenient reporter for real-time or end-point readout on a well plate reader for new adopters of this method. For new protein sequences, copy the amino acid sequence of the desired protein and paste it into the desired codon optimization tool^61,62. There are usually many available organisms and strains of E. coli in the codon optimization tool, but c.......

Materials

Name	Company	Catalog Number	Comments
Alaline	Formedium	DOC0102
Ammonium glutamate	MP Biomedicals	MP21805951
Arginine	Formedium	DOC0106
Asparagine	Formedium	DOC0114
Aspartic Acid	Formedium	DOC0118
ATP	Sigma	A2383
Axygen Sealing Film	Corning	PCR-SP
CMP	Sigma	C1006
Coenzyme A	Sigma	C3144
CutSmart Buffer	NEB	B7204S	Provided with HindIII
Cysteine	Formedium	DOC0122
DNA Clean and Concentrator Kit	Zymo Research	D4004	Used for purifying DNA
dNTPs	NEB	N0447
E. coli tRNA	Sigma (Roche)	10109541001
Folinic Acid	Sigma	47612
Gene Fragment	IDT
Glutamic Acid	Formedium	DOC0134
Glutamine	Formedium	DOC0130
Glycine	Formedium	DOC0138
GMP	Sigma	G8377
HEPES	Sigma	H3375
HindIII-HF	NEB	R3104L
Histidine	Formedium	DOC0142
Isoleucine	Formedium	DOC0150
Leucine	Formedium	DOC0154
Lysine	Formedium	DOC0158
Magnesium glutamate	Sigma	49605
Methionine	Formedium	DOC0166
Microtiter Plate (384 well)	Greiner	781906
Microtiter Plate (96 well)	Greiner	655809
Multimode Plate Reader	BioTek	Synergy Neo2
NAD	Sigma	N8535
NanoPhotometer	Implen	NP80
OneTaq DNA Polymerase	NEB	M0480
PCR Tube	VWR	20170-012
Phenylalanine	Formedium	DOC0170
Phosphoenolpyruvate	Sigma (Roche)	10108294
Potassium glutamate	Sigma	G1501
Potassium oxalate	Fisher Scientific	P273
Proline	Formedium	DOC0174
Putrescine	Sigma	P5780
Serine	Formedium	DOC0178
Spermidine	Sigma	S0266
T4 DNA Ligase	NEB	M0202S
T4 DNA Ligase Reaction Buffer	NEB	B0202S	Provided with T4 DNA Ligase
TempliPhi Amplification Kit	Cytiva	25640010	Used for RCA
Thermal Cycler	Biorad	C1000 Touch
Thermoblock	Eppendorf	ThermoMixer FP
Threonine	Formedium	DOC0182
Tryptophan	Formedium	DOC0186
Tyrosine	Formedium	DOC0190
UMP	Sigma	U6375
Valine	Formedium	DOC0194