Blue Light-inducible Semi-random Mutagenesis on Specific Exogenous DNA in Escherichia coli BL21 Strain

Summary

The outlined protocol describes streamlined methods for recognizing any exogenous DNA while generating mutations only on those exogenous DNAs in Escherichia coli.

Abstract

Mutagenesis technologies have been widely used in variation breeding, directed evolution, and protein engineering. However, most methods can only induce random genomic DNA mutations that are uncontrollable, leading to deformities or even lethal effects. Here, by leveraging the fusion of three systems: cytosine base editor (CBE), blue light-inducible p-Mag/n-Mag elements, and split T7 RNA polymerase, this outlined method incorporates a blue-light-controllable, DNA region-specific, semi-random mutagenesis system.

This system can mutate cytosine to thymine on DNAs starting from the T7 promoter and ending in the T7 terminator. Any exogenous gene downstream of the T7 promoter can be potentially mutated. These tools were also designed in two parts to make the system blue-light-controllable. The first part was created by fusing CBE with n-Mag and the N-terminal-T7 RNA polymerase gene. The second part involved fusing p-Mag with the C-terminal-T7 RNA polymerase. Under blue light, these two parts were assembled to function as a mutation generator on DNAs between the T7 promoter and the T7 terminator. Without blue light, p-Mag is detached from n-Mag, releasing the CBE from editing the DNA.

The aim of this protocol is to generate region-specific random mutations in Escherichia coli with blue light control and further validate the mutated plasmids. The system can be adapted to an in vivo evolution system where the function of the gene of interest can be selected, and the desired mutations can be collected for further study.

Introduction

Random mutation and natural selection are the key forces driving evolution, which has generated large genetic variation across species. The evolutionary process has been utilized as a powerful tool to engineer proteins with optimized or novel functions¹. Natural mutation rates from DNA replication have been estimated to be about one in 10⁹ bases per cell division in multiple prokaryotes and eukaryotes². Low natural mutation rates make it impractical to generate large genetic diversity in laboratories. Studies have focused on developing methods that increase the mutation rates to cover as many sites as possible....

Protocol

The following protocol outlines how to perform exogenous DNA mutagenesis design using CBE-Mag-T7 polymerase mutagenesis system (Step 1). Once the exogenous DNA plasmid is constructed, steps for transformation of the two plasmids are described for expressing CBE-Mag-T7 polymerase mutagenesis in the E. coli BL21 strain by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) and followed by irradiating with blue light forinitiating the mutagenesis (Steps 2-9.2). The quality control steps for evaluating how many mutations were generated on either GFP or exogenous DNA were performed using Sanger sequencing (Steps 9.3-11.2).

Results

The methods outlined in this protocol are for the creation of mutations on exogenous DNA using a split CBE-Mag-T7 RNA polymerase mutation generator. Genes for building the CBE-Mag-T7 RNA polymerase mutation system and the target gene for mutagenesis are all incorporated into the pET-11a plasmid backbone. Successful transformation and blue light irradiation were performed without any damage to the bacterial genomic DNA, shown through normal and comparable colony numbers on LB-agar (Figure 2)........

Discussion

The polymerase-guided base editing tool was first created by Cravens et al. in 2021, using a protein fusion strategy¹⁷. Mutations on four DNA nucleotides were successfully incorporated at rates greater than 10^-4 mutations per bp. However, since mutations were continuously introduced between T7 promotor and terminator without additional control by researchers¹⁸, "good" mutations could not be preserved from ongoing editing. To address this limitation, we in.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
50 mL Conical Centrifuge Tubes	Falcon	CLS352070
Ampicillin	Thermo Scientific Chemicals	J60977.14
Blue-light irradiator	Thorlabs	LIU470A
DNA Assembly Cloning Kit	NEB	E5520S
DNA/Prmers synthesis	BGI Genomics
EndoFree Plasmid Midi Kit	CWBIO	CW2106S	Buffer P1: resuspension buffer, Buffer P2: lysis buffer, Buffer E3: equilibration buffer, Buffer PS: neutralization buffer, Buffer PW: wash buffer,Endo-Free Buffer EB: DNA purification buffer,RNase A Endo-Remover FM: endotoxin removal buffer, Spin Columns DM with Collection Tubes
Glycerol	Fisher Chemical	G33-4
Incubated Shaker	Thermo Scientific	SK2001CPKG
IPTG (isopropylthio-β-galactoside)	Invitrogen	15529019
Microplate Reader	Thermo Scientific	Varioskan LUX
Microscope	Leica	DMI 4000B
Multiskan FC	Thermo Scientific Chemicals
One Shot BL21(DE3) Chemically Competent E. coli	Invitrogen	C600003
PureLink Fast Low-Endotoxin Midi Plasmid Purification Kit	Invitrogen	A36227
Pyrex heavy-duty round-bottom centrifuge tubes, with phenolic screw cap and rubber liner	Corning 8422	CLS8422100