Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

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

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 detection1,2,3,4,5,6, micronutrient and small molecule detection7,8,9,10,11,12, biomanufacturing13,14

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&#.......

Representative Results

Expression of sfGFP from RCA templates was comparable to that of the pJL1 plasmid when using only 0.30 µL of unpurified RCA DNA in a 15 µL reaction (Figure 2A). In fact, doubling and tripling the amount of template appears to offer no benefit in BL21 DE3 Star extract, suggesting already saturated levels of the template at 0.30 µL per reaction. Conversely, there appears to be a benefit to increasing the amount of RCA template when added to cell extract sourced from the SHuffle .......

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 tool61,62. There are usually many available organisms and strains of E. coli in the codon optimization tool, but c.......

Acknowledgements

The authors acknowledge NIH 1R35GM138265-01 and NSF 2029532 for partial support of this project.

....

Materials

NameCompanyCatalog NumberComments
AlalineFormediumDOC0102
Ammonium glutamateMP BiomedicalsMP21805951
ArginineFormediumDOC0106
AsparagineFormediumDOC0114
Aspartic AcidFormediumDOC0118
ATPSigmaA2383
Axygen Sealing FilmCorningPCR-SP
CMPSigmaC1006
Coenzyme ASigmaC3144
CutSmart BufferNEBB7204SProvided with HindIII
CysteineFormediumDOC0122
DNA Clean and Concentrator KitZymo ResearchD4004Used for purifying DNA
dNTPsNEBN0447
E. coli tRNASigma (Roche)10109541001
Folinic AcidSigma47612
Gene FragmentIDT
Glutamic AcidFormediumDOC0134
GlutamineFormediumDOC0130
GlycineFormediumDOC0138
GMPSigmaG8377
HEPESSigmaH3375
HindIII-HFNEBR3104L
HistidineFormediumDOC0142
IsoleucineFormediumDOC0150
LeucineFormediumDOC0154
LysineFormediumDOC0158
Magnesium glutamateSigma49605
MethionineFormediumDOC0166
Microtiter Plate (384 well)Greiner781906
Microtiter Plate (96 well)Greiner655809
Multimode Plate ReaderBioTekSynergy Neo2
NADSigmaN8535
NanoPhotometerImplenNP80
OneTaq DNA PolymeraseNEBM0480
PCR TubeVWR20170-012
PhenylalanineFormediumDOC0170
PhosphoenolpyruvateSigma (Roche)10108294
Potassium glutamateSigmaG1501
Potassium oxalateFisher ScientificP273
ProlineFormediumDOC0174
PutrescineSigmaP5780
SerineFormediumDOC0178
SpermidineSigmaS0266
T4 DNA LigaseNEBM0202S
T4 DNA Ligase Reaction BufferNEBB0202SProvided with T4 DNA Ligase
TempliPhi Amplification KitCytiva25640010Used for RCA
Thermal CyclerBioradC1000 Touch
ThermoblockEppendorfThermoMixer FP
ThreonineFormediumDOC0182
TryptophanFormediumDOC0186
TyrosineFormediumDOC0190
UMPSigmaU6375
ValineFormediumDOC0194

References

Explore More Articles

Cell free SystemsProtein PrototypingRolling Circle AmplificationPCRDNA TemplateRestriction DigestionDNA LigationEnzymatic Amplification

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved