Financial support for this research was provided by startup funds from the University of California.

1. Preparation of bacterial cultures
<ol>
	<li>Grow E. coli strain MG1655 with plasmid transposon constructs (previously described in Kim et al.8) overnight in LB with the appropriate antibiotics (25 &#181;g/mL of kanamycin, see Table of Materials) at 37 &#176;C. 
	NOTE: The sequences of the constructs used and the related sequences are available as GenBank20 accession numbers OP581959, OP581957, OP581958, OP717084, and OP71...

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposable+elements+as+mutator+genes+in+evolution.">Transposable elements as mutator genes in evolution.</a> Nature. 303 (5918), 633-635 (1983).</li><li>Coufal, N. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L1+retrotransposition+in+human+neural+progenitor+cells.">L1 retrotransposition in human neural progenitor cells.</a> Nature. 460 (7259), 1127-1131 (2009).</li><li>Kano, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L1+retrotransposition+occurs+mainly+in+embryogenesis+and+creates+somatic+mosaicism.">L1 retrotransposition occurs mainly in embryogenesis and creates somatic mosaicism.</a> Genes &amp; Development. 23 (11), 1303-1312 (2009).</li><li>Belancio, V. P., Deininger, P. L., Roy-Engel, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LINE+dancing+in+the+human+genome:+transposable+elements+and+disease.">LINE dancing in the human genome: transposable elements and disease.</a> Genome Medicine. 1 (10), 97 (2009).</li><li>Goodier, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrotransposition+in+tumors+and+brains.">Retrotransposition in tumors and brains.</a> Mobile DNA. 5, 11 (2014).</li><li>Kim, N. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+transposable+element+activity+in+individual+live+cells.">Real-time transposable element activity in individual live cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (26), 7278-7283 (2016).</li><li>Lutz, R., Bujard, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+and+tight+regulation+of+transcriptional+units+in+Escherichia+coli+via+the+LacR/O,+the+TetR/O+and+AraC/I1-I2+regulatory+elements.">Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements.</a> Nucleic Acids Research. 25 (6), 1203-1210 (1997).</li><li>Calos, M. P., Miller, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA+sequence+change+resulting+from+the+IQ1+mutation,+which+greatly+increases+promoter+strength.">The DNA sequence change resulting from the IQ1 mutation, which greatly increases promoter strength.</a> Molecular and General Genetics MGG. 183 (3), 559-560 (1981).</li><li>Markwardt, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+cerulean+fluorescent+protein+with+enhanced+brightness+and+reduced+reversible+photoswitching.">An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching.</a> PLoS One. 6 (3), 17896 (2011).</li><li>Nagai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variant+of+yellow+fluorescent+protein+with+fast+and+efficient+maturation+for+cell-biological+applications.">A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.</a> Nature Biotechnology. 20 (1), 87-90 (2002).</li><li>Kersulyte, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposable+element+ISHp608+of+Helicobacter+pylori:+nonrandom+geographic+distribution,+functional+organization,+and+insertion+specificity.">Transposable element ISHp608 of Helicobacter pylori: nonrandom geographic distribution, functional organization, and insertion specificity.</a> Journal of Bacteriology. 184 (4), 992-1002 (2002).</li><li>Shmakov, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+and+evolution+of+class+2+CRISPR-Cas+systems.">Diversity and evolution of class 2 CRISPR-Cas systems.</a> Nature Reviews Microbiology. 15 (3), 169-182 (2017).</li><li>Bao, W., Jurka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homologues+of+bacterial+TnpB_IS605+are+widespread+in+diverse+eukaryotic+transposable+elements.">Homologues of bacterial TnpB_IS605 are widespread in diverse eukaryotic transposable elements.</a> Mobile DNA. 4 (1), 12 (2013).</li><li>Siguier, P., Gourbeyre, E., Chandler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+insertion+sequences:+their+genomic+impact+and+diversity.">Bacterial insertion sequences: their genomic impact and diversity.</a> FEMS Microbiology Reviews. 38 (5), 865-891 (2014).</li><li>Altae-Tran, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+widespread+IS200/IS605+transposon+family+encodes+diverse+programmable+RNA-guided+endonucleases.">The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases.</a> Science. 374 (6563), 57-65 (2021).</li><li>Karvelis, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon-associated+TnpB+is+a+programmable+RNA-guided+DNA+endonuclease.">Transposon-associated TnpB is a programmable RNA-guided DNA endonuclease.</a> Nature. 599 (7886), 692-696 (2021).</li><li>Kaur, D., Kuhlman, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IS200/IS605+family-associated+TnpB+increases+transposon+activity+and+retention.">IS200/IS605 family-associated TnpB increases transposon activity and retention.</a> bioRxiv. , (2022).</li><li>Benson, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GenBank.">GenBank.</a> Nucleic Acids Research. 41, 36-42 (2013).</li><li>Shaner, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+monomeric+red,+orange+and+yellow+fluorescent+proteins+derived+from+Discosoma+sp.+red+fluorescent+protein.">Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.</a> Nature Biotechnology. 22 (12), 1567-1572 (2004).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Ton-Hoang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Stranded+DNA+transposition+is+coupled+to+host+replication.">Single-Stranded DNA transposition is coupled to host replication.</a> Cell. 142 (3), 398-408 (2010).</li><li>Wang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+growth+of+Escherichia+coli.">Robust growth of Escherichia coli.</a> Current Biology. 20 (12), 1099-1103 (2010).</li></ol>

The authors declare no conflicts of interest.

The unique method presented here for real-time imaging of transposable element activity in live cells is a sensitive assay that can directly detect transposition in live cells and in real-time and correlate this activity with the expression of accessory proteins. While the throughput is lower than can be accomplished by bulk methods, this method achieves detailed measurements of TE activity and protein expression in individual living cells.
A variety of tools and techniques can be employed to ...

Transposable elements (TEs) are genetic elements that mobilize within their host genomes by excision or catalyze copying followed by genomic reintegration. TEs exist in all domains of life, and transposition restructures the host genome, mutating coding and control regions1. This generates mutations and diversity that play an important role in evolution2,3, development4,5, and several human diseases6, including cancer7.
Using novel genetic constructs that couple aspects of transpositional activity to fluorescent reporters, our previous work described the development of an experimental system based on the bacterial TE IS608, a representative of the widespread IS200/IS605 family of TEs, that allows for the real-time visualization of transposition in individual live cells8 (Figure 1). The TE system is displayed in Figure 1A. The TE comprises the transposase coding sequence, tnpA, flanked by Left End (LE) and Right End (RE) imperfect palindromic repeats (IPs), which are the recognition and excision sites for TnpA. tnpA is expressed using the promoter PLTetO1, which is repressed by the tet repressor and is inducible with anhydrotetracycline (aTc)9. The TE splits the -10 and -35 sequences of a constitutive PlacIQ1 promoter10 for the blue reporter mCerulean311. As shown in Figure 1C, when the production of tnpA is induced, the TE can be excised, leading to promoter reconstitution. The produced cell expresses mCerulean3 and fluoresces blue. The N-terminus of TnpA is fused to the yellow reporter Venus12, allowing measurement of the TnpA levels by yellow fluorescence.
IS608 and other members of the IS200/IS605 family of transposons also typically encode a second gene of the thus far unknown function, tnpB13. The TnpB proteins are a tremendously abundant but imperfectly characterized family of nucleases encoded by several bacterial and archaeal TEs14,15, which often consist of only tnpB16. Furthermore, recent studies have renewed interest in TnpB by finding that TnpB functions as a CRISPR/Cas-like programmable RNA-guided endonuclease that will yield either dsDNA or ssDNA breaks under diverse conditions17,18. However, it remains unclear what role TnpB may play in regulating transposition. To perform real-time visualization of the effects of TnpB on IS608 transposition, a version of the transposon, including the coding region of TnpB with an N-terminal fusion to the red fluorescent protein mCherry, was created.
Complementing more detailed bulk-level studies performed by the Kuhlman lab19, it is shown here how real-time imaging of transposon activity can quantitatively reveal the impact of TnpB or any other accessory proteins on transpositional dynamics. By fusing TnpB to mCherry, the individual transpositional events are identified by blue fluorescence and correlated with expression levels of TnpA (yellow fluorescence) and TnpB (red fluorescence).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2 Ton Clear Epoxy</td>
			<td>Devcon</td>
			<td>31345</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>5066</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>AX1385-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrotetracycline hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>37919</td>
			<td></td>
		</tr>
		<tr>
			<td>Argon Laser</td>
			<td>Melles Griot</td>
			<td>35-IMA-840-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue Filter Cube</td>
			<td>Chroma</td>
			<td>Ex: Z457/10X, Em: ET485/30M</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse Ti-E Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>Discontinued</td>
		</tr>
		<tr>
			<td>Eppendorf epTIPS Boxes and Refill Trays, Volume: 0.1 to 10 &#181;L, Length: 3.4 cm, 1.33 in., PP (Polypropylene)</td>
			<td>Eppendorf North America Biotools</td>
			<td>22491504</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf epTIPS Boxes and Refill Trays, Volume: 50 to 1000 &#181;L, Length: 7.1 cm, 2.79 in., PP (Polypropylene)</td>
			<td>Eppendorf North America Biotools</td>
			<td>22491555</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferrous Sulfate Acs 500 g</td>
			<td>Fisher Scientific</td>
			<td>706834</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td></td>
			<td></td>
			<td>Fiji (imagej.net)</td>
		</tr>
		<tr>
			<td>Fisher BioReagents&#160;LB Broth, Miller (Granulated)</td>
			<td>Fisher Scientific</td>
			<td>BP9723-2&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Cover Slide</td>
			<td>Fisher Scientific</td>
			<td>12-542B&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>1355006</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate Cert Ac</td>
			<td>Fisher Scientific</td>
			<td>XXM63SP3KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Heater</td>
			<td>World Precision Instruments</td>
			<td>96810-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>Fisher Scientific</td>
			<td>17001H</td>
			<td></td>
		</tr>
		<tr>
			<td>ProScan III Stage</td>
			<td>Prior</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Red Filter Cube</td>
			<td>Chroma</td>
			<td>Ex: ET560/40X, Em: ET645/75M</td>
			<td></td>
		</tr>
		<tr>
			<td>Sapphire 561 LP Laser</td>
			<td>Coherent</td>
			<td>1170412</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide, Microscope</td>
			<td>Fisher Scientific</td>
			<td>125535B</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiamine Hydrochloride</td>
			<td>Sigma-Aldrich (SIAL)</td>
			<td>T1270-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti-LU4 Laser Launch</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Yellow Filter Cube</td>
			<td>Chroma</td>
			<td>Ex: Z514/10X, Em: ET535/30M</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time quantification of the effects of is200/is605 family-associated tnpb on transposon activity

Here, a protocol is outlined to perform live, real-time imaging of transposable element activity in live bacterial cells using a suite of fluorescent reporters coupled to transposition. In particular, it demonstrates how real-time imaging can be used to assess the effects of the accessory protein TnpB on the activity of the transposable element IS608, a member of the IS200/IS605 family of transposable elements. The IS200/IS605 family of transposable elements are abundant mobile elements connected with one of the most innumerable genes found in nature, tnpB. Sequence homologies propose that the TnpB protein may be an evolutionary precursor to CRISPR/Cas9 systems. Additionally, TnpB has received renewed interest, having been shown to act as a Cas-like RNA-guided DNA endonuclease. The effects of TnpB on the transposition rates of IS608 are quantified, and it is demonstrated that the expression of TnpB of IS608 results in ~5x increased transposon activity compared to cells lacking TnpB expression.

This method of visualizing transposon activity in live cells by fluorescence microscopy, while having lower throughput than bulk fluorescence measurements, allows direct visualization of transposon activity in individual live cells. Transposon excision events result in the reconstitution of the promoter for mCerulean3 (Figure 1), allowing identification of cells undergoing transposon activity by bright blue fluorescence (Figure 2, TnpB+: Supplementary Mo...

Watch this Scientific Journal Video about Real-Time Quantification of the Effects of IS200/IS605 Family-Associated TnpB on Transposon Activity at JoVE.com

Real-Time Quantification of the Effects of IS200/IS605 Family-Associated TnpB on Transposon Activity

A protocol is outlined to perform live real-time imaging to quantify how the accessory protein TnpB affects the dynamics of transposition in individual live Escherichia coli cells.

Here, a protocol is outlined to perform live, real-time imaging of transposable element activity in live bacterial cells using a suite of fluorescent ...

This protocol allows us to quantify excision statistics on a single-cell basis, as opposed to looking at bulk behavior. At this level, we can track the behavior of individual cells over time, including their TnpA and TnpB levels for cells that excise versus cells that don't, which can allow us to, for example, measure the effects of identified mutational events on growth rate. This method is a simple, low-cost way to measure live cell mutational dynamics.The general approach is applicable to any system that requires live cell fluorescent imaging. Prepare a slide by boiling M63 with 0.5%glucose and 1.5%agarose in the microwave to melt the agarose. Ensure that the medium is completely melted and well-mixed.Allow the mixture to cool to 55 degrees Celsius before adding antibiotics and inducers. Place a microscope slide on the workbench. Stack one slide perpendicular to the first.Ensure that there is a gap equal to one slide thickness between the bottom and top slide. Pipette one milliliter of the M63 agarose mixture into the gap between the slide slowly to create a small gel square. Once the gel has solidified, slide the top slide to remove it.Trim the agarose pad with a razor blade or knife. Then, pipette 2.5 microliters of the bacterial culture and put the cover slip on top. Seal the space between the slide and the cover slip with epoxy.Allow the epoxy to dry and the cells to settle onto the agarose pad for one hour at 37 degrees Celsius. Place the prepared sample on a fluorescence microscope in an environment heated and maintained at 37 degrees Celsius. For each wavelength, find the field-of-view containing minimal fluorescence.Set up a protocol to acquire images in a grid at different wavelengths and at regular time intervals. Transposon excision events result in the reconstitution of the promoter for mCerulean3, allowing the identification of cells undergoing transposon activity by bright blue fluorescence. TnpB enhances transposon excision rate.Cells expressing accessory protein TnpB experience four to five times higher levels of transposon activity compared to TnpB-negative cells. In TnpB-positive cells, cells undergoing transposon excision events have higher expression levels of mCherry TnpB than the general population. Moreover, for cells undergoing excision events, TnpB-positive cells express only marginally higher levels of venous TnpA-transposase than TnpB-negative cells, which is higher than the yellow fluorescence of the general population.The most likely place to struggle is in the preparation of the cell culture for the experiment. You should make sure cells are grown to exponential phase and that the slide is inoculated with an appropriate number of cells. Remember to watch out for bubbles when sealing the slide so the agarose gel does not dry out.

real-time-quantification-effects-is200is605-family-associated-tnpb-on

Research

JoVE Journal

Biology

2.2K vistas.  University of California. Este protocolo nos permite cuantificar las estadísticas de escisión sobre una sola célula, en lugar de observar el comportamiento masivo. En este nivel, podemos rastrear el comportamiento de las células individuales a lo largo del tiempo, incluidos sus niveles de TnpA y TnpB para las células que se extirpan frente a las células que no lo hacen, lo que puede permitirnos, por ejemplo, medir los efectos de los eventos mutacionales identificados en la tasa de crecimiento. Este método es un...