Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Summary

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

Abstract

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Introduction

The function of any genetic element can be affected by its location in the genome. In bacteria, this mainly results from interference by the transcription of neighboring genes, local DNA topology, and/or replication-associated gene dosage. In particular, the processes of DNA replication and segregation are controlled, at least in part, by non-coding chromosomal regions¹, and the proper function of these regions depends on genomic location/context. In E.coli, examples are the dif site, required for sister chromosome resolution²; KOPS sequences, required for chromosome segregation³....

Protocol

1. Collection of the Transposon Library

NOTE: The chromosomal DARS2 locus was cloned into the mini Tn10-based transposon, NKBOR (on pNKBOR)²¹, resulting in NKBOR::DARS2 (pJFM1). pNKBOR can be obtained online²². pNKBOR is a R6K-based suicide vector that requires the initiator protein π for replication²³. Plasmid pJFM1 is therefore able to replicate in an E. coli strain (e.g., Dh5α λ pir) containing a chromosomal copy of the pir gene. However, when pJFM1 is transformed into the Pir-deficient wildtype MG165....

Results

A Southern blot was done to verify that DARS2 was distributed randomly throughout the chromosome in the transposon library (t = 0) and that the fittest clones would persist over time. The Southern blot was performed on DNA extracted from the initial transposon pool (at t = 0) and every estimated 100 out of 700 generations of competition (Figure 3). Here, the total cellular DNA from each time-point was digested with the PvuI restriction enzym.......

Discussion

The methodology used here takes advantage of state-of-the-art techniques to answer a difficult question regarding the optimal genomic position of a genetic element. The random insertion of the genetic element (mediated by the transposon) enables the fast and easy collection of thousands of clones, which then can be made to compete against each other to select for the optimal position of the investigated genetic element (i.e., the fittest clone).

Here, DARS2 was inserted into .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Autoclaved Mili-Q water		None
Electroporation Cuvettes, 0.1 cm	Thermo Fisher Scientific	P41050
Bio-Rad MicroPulser Electroporation System	Bio-Rad	165-2100
LB Broth	Thermo Fisher Scientific	12780029
LB Agar, powder (Lennox L agar)	Thermo Fisher Scientific	22700025
Glycerol	Thermo Fisher Scientific	17904
Fisherbrand Plastic Petri Dishes	Fisher Scientific	S33580A
Falcon 50mL Conical Centrifuge Tubes	Fisher Scientific	14-432-22
Falcon 15mL Conical Centrifuge Tubes	Fisher Scientific	14-959-53A
Nunc CryoTubes	Sigma-Aldrich	V7634
Phusion High-Fidelity DNA Polymerase (2 U/µL)	Thermo Fisher Scientific	F530S
dATP, [α-32P]- 3000Ci/mmol 10mCi/ml, 250 µCi	PerkinElmer	BLU012H250UC
DECAprime II DNA Labeling Kit	Thermo Fisher Scientific	AM1455
Spectrophotometer  SF/MBV/03.32	Pharmacia
Hermle Centrifuge    SF/MBV/03.46	Hermle
Ole Dich Centrifuge  SF/MBV/03.29	Ole Dich
Eppendorftubes 1.5 mL	Sigma-Aldrich	T9661
Eppendorftubes 2.0 mL	Sigma-Aldrich	T2795
Sodium Chloride	Merck	6404
96% Ethanol	Sigma-Aldrich	16368
Trizma HCl	Sigma-Aldrich	T-3253
Phenol Ultra Pure	BRL	5509UA
Chloroform	Merck	2445
Ribonuclease A type II A	Sigma-Aldrich	R5000
Sodiumdodecylsulphate (SDS)	Merck	13760
Lysozyme	Sigma-Aldrich	L 6876
Isopropanol	Sigma-Aldrich	405-7
0.5M Na-EDTA pH 8.0	BRL	5575 UA
Kanamycin sulfate	Sigma-Aldrich	10106801001
PvuI (10 U/µL)	Thermo Fisher Scientific	ER0621
UltraPure Agarose	Thermo Fisher Scientific	16500500
DNA Gel Loading Dye (6X)	Thermo Fisher Scientific	R0611
Tris-Borate-EDTA buffer	Sigma-Aldrich	T4415
Ethidium bromide	Sigma-Aldrich	E7637
Hydrochloric acid	Sigma-Aldrich	433160
Sodium Hydroxide	Sigma-Aldrich	71687
Whatman 3MM papers	Sigma-Aldrich	WHA3030931
SSC Buffer 20× Concentrate	Sigma-Aldrich	S6639
Amersham Hybond-N+	GE Healthcare	RPN119B
Ficoll 400	Sigma-Aldrich	F8016
Polyvinylpyrrolidone	Sigma-Aldrich	PVP40
Bovine Serum Albumin - Fraction V	Sigma-Aldrich	85040C
Deoxyribonucleic acid sodium salt from salmon testes	Sigma-Aldrich	D1626
Carestream Kodak  BioMax  light film	Sigma-Aldrich	Z373494
GenElute  Gel Extraction Kit	Sigma-Aldrich	NA1111
GenElute  PCR Clean-Up Kit	Sigma-Aldrich	NA1020
T100  Thermal Cycler	Bio-Rad
SmartSpec Plus Spectrophotometer	Bio-Rad
Rifampicin	Serva	34514.01
Cephalexin	Sigma-Aldrich	C4895
Mithramycin	Serva	29803.02
Magnesium chloride hexahydrate	Sigma-Aldrich	246964
Apogee A10 instrument	Apogee