Genome-wide Gene Deletioner i<em> Streptococcus sanguinis</em> Av High Throughput PCR

Summary

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Abstract

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria ^1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5′ end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Protocol

1. Primer Design Primers are designed using in house scripts based on the S. sanguinis SK36 genome sequence. Three sets of primers, F1/R1, F2/R2, and F3/R3 are designed for amplification of the 1-kb upstream sequence of the target gene, the aphA-3 gene encoding kanamycin resistance (Kmr) protein3 and the 1-kb downstream sequence of the target gene, respectively (Figure 1). Among these primers, F1 and R3 are designed using ePrimer3 in the EMBOSS suite of…

Representative Results

After PCR amplification using primers F1 and R1, and F3 and R3, approximately 1-kb upstream and downstream of each S. sanguinis gene were obtained in 96-well format, respectively (Figure 2A). Under our PCR conditions and using designed primers, a specific product was amplified from S. sanguinis genomic DNA in each PCR reaction. This result indicated the primers were highly specific to the targets of S. sanguinis. Through PCR re-amplification using primers F1 and R3 and t…

Discussion

To minimize the possible polar effects of the replacement of one targeted gene with exogenous antibiotics gene on neighboring genes, two steps are taken while initial primer designing. For most of deleted genes, the primers R1 and F3 are designed to delete the coding region from 6 bp following the start codon to 30 bp prior to the stop codon. The last 30 base pairs are retained to protect potential ribosomal binding site used by the adjacent downstream gene. The retained region between two neighboring genes is extended t…

Materials

Names	Company	Catalog#	Comments (optional)
Primer F2	Integrated DNA Technologies		TGACTAACTAGGAGGAATAAATG GCTAAAATGAGAATAT
Primer R2	ibid		CATTATTCCCTCCAGGTACTAAAA CAATTCATCCAGT
Primer F2p	ibid		GATAAACCCAGCGAACCATTTGA
Primer P1	ibid		GCTTATATACCTTAGCAGGAGACA
Primer P2	ibid		GTATGACATTGCCTTCTGCGTCC
Primer 5′ end_R1_seq	ibid		GCCATTTATTCCTCCTAGTTAGTCA
Primer 5′ end_F3_seq	ibid		GTTTTAGTACCTGGAGGGAATAATG
Primer 5′ end_R1p_seq	ibid		CCTCAAATGGTTCGCTGGGTTTATC
CSP	Synprep		Sequence:NH2-DLRGVPNPWGWIFGR-COOH
DNA Polymerase	Invitrogen	11304-102	Platinum Taq DNA Polymerase High Fidelity
EcoRI	New England Biolabs Inc	R0101S	Restriction enzyme
Agar	American Bioanalytical	AB01185
Agarose	Promega	V3125
BHI	BD Biosciences	237500	Brain Heart Infusion
Horse serum	Fisher Scientific	SH3007403	Horse serum
Km	Fisher Scientific	BP906-5	Kanamycin
TH broth	BD Biosciences	249240	Todd Hewitt broth
PureLink 96	Invitrogen	K310096	PureLink 96 PCR purification kit
QIAquick	Qiagen	28106	QIAquick PCR purification kit
Filter	Corning	431117	0.22 μm polystyrene filter
Anoxomat System	Mart Microbiology b.v.	Anoxomat Mark II
Benchtop centrifuge	Thermo Scientific	75004377	Sorvall Legend RT Plus microplate rotor
Gel Documentation	UVP LLC	BioDoc-It 210
Incubator	Fisher Scientific	11-690-625D	Isotemp
Labnet’s Gel XL	Labnet	E0160	Labnet’s Gel XL
Microcentrifuge	Eppendorf	22621408	Microcentrifuge 5415 R
Multichannel pipettes	Thermo Scientfic	4661040, 4661020	Finnpipette F1
Thermal Cycler	Applied Biosystems Inc.	N805-0200	GeneAmp PCR system 9700