Name: determination of the optimal chromosomal location(s) for a dna element in escherichia coli using a novel transposon-mediated approach
Uploaded: 2017-09-11T13:00:00
Description: 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

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., ...

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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.

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			<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>
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		<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>
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			<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>
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		<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>
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		<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>
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			<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>
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			<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>
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		</tr>
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			<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">
			<td height="20" style="width:369px;height:20px;">Spectrophotometer&#160; SF/MBV/03.32</td>
			<td style="width:164px;">Pharmacia</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Hermle Centrifuge&#160;&#160;&#160; SF/MBV/03.46</td>
			<td style="width:164px;">Hermle</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Ole Dich Centrifuge&#160; SF/MBV/03.29</td>
			<td style="width:164px;">Ole Dich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Eppendorftubes 1.5 mL</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">T9661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Eppendorftubes 2.0 mL</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">T2795</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Merck</td>
			<td>6404</td>
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		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">96% Ethanol</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">16368</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Trizma HCl</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">T-3253</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Phenol Ultra Pure</td>
			<td>BRL</td>
			<td>5509UA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Chloroform</td>
			<td>Merck</td>
			<td>2445</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Ribonuclease A type II A</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td>R5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Sodiumdodecylsulphate (SDS)</td>
			<td>Merck</td>
			<td>13760</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Lysozyme</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td>L 6876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Isopropanol</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td>405-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">0.5M Na-EDTA pH 8.0</td>
			<td>BRL</td>
			<td>5575 UA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Kanamycin sulfate</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">10106801001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:369px;height:42px;">PvuI (10 U/&#181;L)</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">ER0621</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:369px;height:42px;">UltraPure Agarose</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">16500500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:369px;height:42px;">DNA Gel Loading Dye (6X)</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">R0611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Tris-Borate-EDTA buffer</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">T4415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Ethidium bromide</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">E7637</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Hydrochloric acid</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">433160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Sodium Hydroxide</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">71687</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Whatman&#160;3MM papers</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">WHA3030931</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">SSC Buffer 20&#215; Concentrate</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">S6639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Amersham Hybond-N+</td>
			<td style="width:164px;">GE Healthcare</td>
			<td style="width:119px;">RPN119B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Ficoll&#160;400</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">F8016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Polyvinylpyrrolidone</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">PVP40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Bovine Serum Albumin - Fraction V</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">85040C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Deoxyribonucleic acid sodium salt from salmon testes</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">D1626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Carestream Kodak &#160;BioMax &#160;light film</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z373494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">GenElute &#160;Gel Extraction Kit</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">NA1111&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">GenElute &#160;PCR Clean-Up Kit</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">NA1020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">T100&#160; Thermal Cycler</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">SmartSpec Plus Spectrophotometer</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Rifampicin</td>
			<td style="width:164px;">Serva</td>
			<td style="width:119px;">34514.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Cephalexin</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">C4895</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Mithramycin</td>
			<td style="width:164px;">Serva</td>
			<td>29803.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Magnesium chloride hexahydrate</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">246964</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Apogee A10 instrument</td>
			<td style="width:164px;">Apogee</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

determination-optimal-chromosomal-locations-for-dna-element

Research

JoVE Journal

Genetics

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. To assess the impact of PM on nuclear genetic integrity, structural chromosomal aberrations are scored in the metaphase spreads of mouse RAW264.7 macrophage cells. PM is collected from ambient air with a high volume total suspended particles sampler. The collected material is solubilized and filtered to retain the water-soluble, fine portion. The particles are characterized for chemical composition by nuclear magnetic resonance (NMR) spectroscopy. Different concentrations of particle suspension are added onto an in vitro culture of RAW264.7 mouse macrophages for a total exposure time of 72 hr, along with untreated control cells. At the end of exposure, the culture is treated with colcemid to arrest cells in metaphase. Cells are then harvested, treated with hypotonic solution, fixed in acetomethanol, dropped onto glass slides and finally stained with Giemsa solution. Slides are examined to assess the structural chromosomal aberrations (CAs) in metaphase spreads at 1,000X magnification using a bright-field microscope. 50 to 100 metaphase spread are scored for each treatment group. This technique is adapted for the detection of structural chromosomal aberrations (CAs), such as chromatid-type breaks, chromatid-type exchanges, acentric fragments, dicentric and ring chromosomes, double minutes, endoreduplication, and Robertsonian translocations in vitro after exposure to PM. It is a powerful method to associate a well-established cytogenetic endpoint to epigenetic alterations.

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter.

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.
This article describes in detail the &#34;Copy Number Ratio of S/G1 for mapping genomic Time of Replication&#34; method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to control gene expression. These regulatory proteins control gene expression either at the level of DNA (transcription) or at the level of RNA (pre-mRNA splicing, polyadenylation, mRNA transport, decay, and translation). Identification of regulatory sequences helps understand not only how a gene is switched on or off, but also which downstream genes are regulated by a particular regulatory protein. Here, we describe a one-step approach that allows saturation mutagenesis of a protein binding site in RNA. It involves doping DNA template with non-wild-type nucleotides within the binding site, synthesis of separate RNAs with each phosphorothioate nucleotide, and isolation of the bound fraction following incubation with protein. Interference from non-wild-type nucleotides results in their preferential exclusion from the protein-bound fraction. This is monitored by gel electrophoresis following selective chemical cleavage with iodine of phosphodiester bonds containing phosphorothioates (phosphorothioate mutagenesis or PTM). This single-step saturation mutagenesis approach is applicable to the characterization of any protein binding site in RNA.

Proteins that bind specific RNA sequences play critical roles in gene expression. Detailed characterization of these binding sites is crucial for our understanding of gene regulation. Here, a single-step approach for saturation mutagenesis of protein-binding sites in RNA is described. This approach is relevant for all protein-binding sites in RNA.

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to ...

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol for transferring gene deletion mutations from one Escherichia coli strain to another by using generalized transduction with the bacteriophage P1. This method requires that the mutation be selectable (e.g., based on gene disruptions using antibiotic cassette insertions). Such antibiotic cassettes can be mobilized from a donor strain and introduced into a recipient strain of interest to quickly and easily generate a gene deletion mutant. The antibiotic cassette can be designed to include flippase recognition sites that allow the excision of the cassette by a site-specific recombinase to produce a clean knock-out with only a ~100-base-pair-long scar sequence in the genome. We demonstrate the protocol by knocking out the tamA gene encoding an assembly factor involved in autotransporter biogenesis and test the effect of this knock-out on the biogenesis and function of two trimeric autotransporter adhesins. Though gene deletion by P1 transduction has its limitations, the ease and speed of its implementation make it an attractive alternative to other methods of gene deletion.

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Here we present a protocol for the use of pre-existing antibiotic resistance-cassette deletion constructs as a basis for making deletion mutants in other E. coli strains. Such deletion mutations can be mobilized and inserted into the corresponding locus of a recipient strain using P1 bacteriophage transduction.

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame (ORF) annotation impose two arbitrary criteria: a minimum length of 100 codons and a single ORF per transcript. However, a growing number of studies report expression of proteins from allegedly non-coding regions, challenging the accuracy of current genome annotations. These novel proteins were found encoded either within non-coding RNAs, 5' or 3' untranslated regions (UTRs) of mRNAs, or overlapping a known coding sequence (CDS) in an alternative ORF. OpenProt is the first database that enforces a polycistronic model for eukaryotic genomes, allowing annotation of multiple ORFs per transcript. OpenProt is freely accessible and offers custom downloads of protein sequences across 10 species. Using OpenProt database for proteomic experiments enables novel proteins discovery and highlights the polycistronic nature of eukaryotic genes. The size of OpenProt database (all predicted proteins) is substantial and need be taken in account for the analysis. However, with appropriate false discovery rate (FDR) settings or the use of a restricted OpenProt database, users will gain a more realistic view of the proteomic landscape. Overall, OpenProt is a freely available tool that will foster proteomic discoveries.

OpenProt is a freely accessible database that enforces a polycistronic model of eukaryotic genomes. Here, we present a protocol for the use of OpenProt databases when interrogating mass spectrometry datasets. Using OpenProt database for analysis of proteomic experiments allows for discovery of novel and previously undetectable proteins.

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device.

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...