The authors were funded by grants from the Novo Nordisk Foundation, the Lundbeck Foundation, and the Danish National Research Foundation (DNRF120) through the Center for Bacterial Stress Response and Persistence (BASP).

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 &#960; for replication23. Plasmid pJFM1 is therefore able to replicate in an E. coli strain (e.g., ...

<ol>
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The authors have no competing financial interest.

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 ...

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; and datA, DARS1, and DARS2 regions, required for proper chromosomal replication control (below; 4). We present a method allowing for the random relocation, selection, and determination of the optimal genetic context of any given genetic element, exemplified here by the study of the DARS2 non-coding region.
In E. coli, DnaA is the initiator protein responsible for DNA strand opening at the single replication origin, oriC, and for the recruitment of the helicase DnaB5,6,7. DnaA belongs to the AAA+ (i.e., ATPases associated with diverse activities) proteins and can bind both ATP and ADP with similar high affinities5. The level of DnaAATP peaks at initiation8, where DnaAATP forms a multimer on oriC that triggers DNA duplex opening9. After initiation, oriC is made temporarily unavailable for re-initiation due to sequestration by a mechanism involving the binding of the SeqA protein to hemimethylated oriC10,11. During sequestration, the level of DnaAATP is reduced by at least two mechanisms: the regulatory inactivation of DnaA (RIDA)12,13 and datA-dependent DnaAATP hydrolysis (DDAH)14,15. Both RIDA and DDAH promote the conversion of DnaAATP to DnaAADP. Prior to a new round of initiation, DnaAADP is re-activated to DnaAATP at specific DnaA-reactivating sequences (DARS): DARS1 and DARS216,17. The chromosomal datA, DARS1,and DARS2 regions are non-coding and act in a chaperone-like manner to modulate DnaAATP/DnaAADP interconversion. These regions, located outside the origin of replication, enable the assembly of a DnaA complex for either the inactivation (datA;14) or activation (DARS1 and DARS2;17) of DnaA. Deleting DARS2 in a cell does not alter mass doubling time but results in asynchronous replication initiation15,16,18. However, DARS2-deficient cells have a fitness cost compared to an otherwise isogenic wildtype during both continuous growth competition in rich medium or during the establishment of colonization in the mouse intestine18. This indicates that even minor changes in asynchrony/origin concentration have a negative effect on bacterial fitness. In E. coli, there is a selective pressure to maintain chromosome symmetry (i.e., two nearly equal length replication arms)19. The datA, DARS1, and DARS2 regions have the same relative distance to oriC in all E. coli strains sequenced18, despite large variations in chromosome size.
Here, we use the DARS2 region of E. coli as an example for the identification of the chromosomal position(s) optimal for its function. DARS2 was inserted into the NKBOR transposon, and the resultant NKBOR::DARS2 transposon subsequently inserted randomly into the genome of MG1655 &#916;DARS2. We thus generated a collection of cells, each possessing DARS2 placed at a different location on the chromosome. An in vitro competition experiment, where all cells in the collection were pooled and competed against each other during continuous growth in LB for an estimated 700 generations, was performed. The outcome of the competition experiment was monitored/determined using Southern blot, easy gene walking, and whole-genome sequencing (WGS; Figure 1). End-point clones resolved by easy gene walking were characterized by flow cytometry to evaluate cell-cycle parameters. In a flow cytometric analysis, cell size, DNA content, and initiation synchrony can be measured for a large number of cells. During flow cytometry, a flow of single cells passes a light beam of the appropriate wavelength to excite the stained DNA, which is then simultaneously registered by photomultipliers that collect the emitted fluorescence, a measure of DNA content, provided the cells are stained for DNA. The forward-scattered light is a measure of cell mass20.
The in vitro competition experiment we present here is used to address questions relating to the importance of the chromosomal position and genomic context of the genetic element. The method is unbiased and easy to use.

<table width="819">
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	<tbody>
		<tr height="20">
			<td height="20" style="width:369px;height:20px;">Autoclaved Mili-Q water</td>
			<td style="width:164px;"></td>
			<td style="width:119px;">None</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:369px;height:20px;">Electroporation Cuvettes, 0.1 cm</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">P41050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:369px;height:20px;">Bio-Rad MicroPulser Electroporation System</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:119px;">165-2100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:369px;height:42px;">LB Broth</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">12780029&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:369px;height:20px;">LB Agar, powder (Lennox L agar)</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">22700025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:369px;height:20px;">Glycerol</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">17904</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Fisherbrand&#160;Plastic Petri Dishes</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:119px;">S33580A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Falcon 50mL Conical Centrifuge Tubes</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:119px;">14-432-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Falcon 15mL Conical Centrifuge Tubes</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:119px;">14-959-53A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Nunc&#160;CryoTubes</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">V7634&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:369px;height:42px;">Phusion High-Fidelity DNA Polymerase (2 U/&#181;L)</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">F530S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">dATP, [&#945;-32P]- 3000Ci/mmol 10mCi/ml, 250 &#181;Ci</td>
			<td style="width:164px;">PerkinElmer</td>
			<td style="width:119px;">BLU012H250UC</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:369px;height:42px;">DECAprime II DNA Labeling Kit</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AM1455</td>
			<td></td>
		</tr>
		<tr height="20">
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determination of the optimal chromosomal location(s) for a dna element in escherichia coli using a novel transposon-mediated approach

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.

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...

Watch this Scientific Journal Video about Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach at JoVE.com

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

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

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

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

The overall goal of this experiment is to determine the optimal chromosomal position of any given DNA element using transposon-mediated random insertion, competition experiments and whole genome sequencing. This method can help answer key questions in the DNA replication field such as the importance of a chromosomal location of a genetic element. The advantage of this technique is that is couples transposon-based random insertion with the selection of the fittest clone by growth advantage.Here, it's simplified by the E.coli DARS2 region. Though this method can provide insight into the chromosomal organization of Escherichia coli, it can also be applied to other bacteria. After preparing electrocompetant cells such as MG1655 delta DARS2 according to the text protocol, add one microgram of pNKBOR DARS2 to 40 microliters of cells and transfer the mixture to a pre-chilled sterile 0.2-centimeter gap cuvette.Insert the cuvette into the electroporation chamber. Then, electroporate the cells at 18 kilovolts, 500 ohm and 25 microfarad. The time constant should be about five milliseconds and no arcing should occur.Quickly recover the bacterial suspension by re-suspending it in one milliliter of pre-warmed LB broth and transfer to a 15-milliliter test tube. Incubate the cells under aerated growth conditions at 37 degrees Celsius for 30 minutes without antibiotic selection. Then, plate the bacteria onto the LB agar plates supplemented with 50 micrograms per milliliter of kanamycin and incubate the plates at 37 degrees Celsius overnight.The following day, count the colonies from the electroporation. One colony is considered equal to one chromosomal transposon insertion. Add one milliliter of LB broth to each plate and wash off all the colonies.Reuse the same one milliliter of LB broth to increase the bacterial concentration. Then, pool all the colonies in the same 50-milliliter tube. Vortex the tube to freeze the starting material and mix one milliliter of cell suspension with one milliliter of 50%glycerol on ice.Transfer the tubes to dry ice for 10 minutes. Once the cultures are frozen, transfer them to a negative 80-degree-Celsius freezer. Thaw the transposon library from negative 80 degrees Celsius on ice.Mix by pipetting, then transfer 100 microliters of the transposon library to 10 milliliters of LB in a 15-milliliter test tube. Grow the cells aerated with continuous shaking at 37 degrees Celsius for eight hours to stationary phase. Adjust the parameters to different growth conditions as desired.To propagate the bacterial population at approximately every 10 generations, transfer 10 microliters of the previously stationary phase into 10 milliliters of fresh pre-warmed medium and grow the culture for another eight hours to stationary phase. After each 100 generations of competition, save five one-milliliter samples and store them in negative 80 degrees Celsius. Prepare the probe for the Southern blot against a one-kilobyte section of the NKBOR by PCR amplification using specific primers as listed here.Run the samples using the following program. Label the resultant PCR fragment with P32-labeled dATP using the random primer system according to Smith and prepare total cellular DNA according to the text protocol. Digest the total cellular DNA with Pvul which cuts NKBOR containing the region of interest outside of a region covered by the probe.Perform a Southern blot using 0.7%agarose gel according to Loebner-Olsen and von Freiesleben. To use easy gene walking to identify DARS2 insertion sites from single clones, after 700 estimated generations of competition, isolate genomic DNA for the PCR template by first spreading bacteria on an LB agar plate and incubate at 37 degrees Celsius overnight. Inoculate LB broth with single colonies and grow them overnight at 37 degrees Celsius.Then, transfer 20 microliters into 200 microliters of autoclaved distilled water. Vortex the colonies into solution, then heat the mixture at 100 degrees Celsius for 10 minutes. Centrifuge the samples at maximum speed for five minutes, then save the cell lysate at negative 20 degrees Celsius for future use.Next, design three nested primers to anneal within the transposon of choice as shown here. Use 200 nanograms of genomic DNA, nested primer one and a random primer to prepare a 20-microliter PCR reaction. Then, run the samples using the following program.For the second amplification, use one microliter of the first reaction as the template and use nested primer two and the same random primer. Carry out a third reaction in a similar manner using nested primer three, the same random primer and one microliter from the second reaction as the template. Run the products from the final PCR reaction on a 1.5%agarose gel and statin with ethidium bromide.Cut out a band in the range of 100 to 800 base pairs and isolate the DNA using any commercial DNA gel extracting kit according to the manufacturer's directions. To carry out flow cytometry, balance each bacterial culture by maintaining it in the exponential growth phase for at least 10 generations. Measure the OD600 and ensure that it never exceeds 0.3.To carry out rifampicin run-out per the text protocol, transfer one milliliter of culture to a 15-milliliter tube containing 30 microliters of RIF-CEF. Incubate the tube at 37 degrees Celsius with a shaking for a minimum of four hours. For the EXP sample, transfer one milliliter of exponentially growing culture to a 1.5 milliliter tube on ice.Then, harvest the cells by centrifugation at 15, 000 times G and four degrees Celsius for five minutes. Re-suspend the pellet in 100 microliters of ice-cold 10 millimolar tris pH 7.5 and add one milliliter of ice-cold 77%ethanol. Store the samples at four degrees Celsius until use.Stain the cells by harvesting 100 to 300 microliters of fixed cells by centrifugation at 15, 000 times G for 15 minutes. Remove the supernatant and use 130 microliters of DNA staining solution to re-suspend the pellet. Then, incubate the samples on ice and in the dark for 10 minutes.Finally, use flow cytometry to determine the origin per cell, the relative cell mass and the relative DNA content. Following the extraction of DNA from the initial transposon pool and every estimated 100 out of 700 generations of competition, the DNA was cut with Pvul which is known to cut transposon NKBOR DARS2 once in a region not covered by the probe and a Southern blot was performed. As shown here, the initial DARS2 pool lacked distinct bands which shows that DARS2 was inserted randomly throughout the chromosome.Over time, a pattern emerged where the initial large pool of DARS2 clones developed into only one or a few persisting DARS2 clones. Whole genome sequencing was used to identify DARS2 insertion sites from the start pool. And after 400, 500, and 700 generations of competition in a representative subset of insertion sequenced, 98%of all DARS2 insertions were found close to the wild type DARS2 chromosomal location.And 2%were found elsewhere on the chromosome. DARS2-deficient cells were previously shown to initiate replication in a synchrony and to have a decrease in origin concentration relative to wild-type cells. Here, the presence of a DARS2 element in the terminus does not restore synchrony or the cellular origin content to wild-type levels while the selected DARS2 clones IR and RPPH do initiate replication in synchrony.After watching this video, you should have a good understanding of how to determine the optimal chromosomal position of a given DNA element. This is done by transposon-based random insertions followed by competition experiments and finally whole genome sequencing.

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JoVE Journal

Genetics

7.4K visualizações.  University of Copenhagen. The overall goal of this experiment is to determine the optimal chromosomal position of any given DNA element using transposon-mediated random insertion, competition experiments and whole genome sequencing. This method can help answer key questions in the DNA replication field such as the importance of a chromosomal location of a genetic element. The advantage of this technique is that is couples transposon-based random insertion with the selection of the fittest clone by growth advantage.