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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 tha....

Protocol

1. Preparation of bacterial cultures

  1. Grow E. coli strain MG1655 with plasmid transposon constructs (previously described in Kim et al.8) overnight in LB with the appropriate antibiotics (25 µg/mL of kanamycin, see Table of Materials) at 37 °C.
    NOTE: The sequences of the constructs used and the related sequences are available as GenBank20 accession numbers OP581959, OP581957, OP581958, OP717084, and OP71.......

Representative Results

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.......

Discussion

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 .......

Acknowledgements

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

....

Materials

NameCompanyCatalog NumberComments
2 Ton Clear EpoxyDevcon31345
AgaroseSigma-Aldrich5066
Ammonium sulfateSigma-AldrichAX1385-1
Anhydrotetracycline hydrochlorideSigma-Aldrich37919
Argon LaserMelles Griot35-IMA-840-015
Blue Filter CubeChromaEx: Z457/10X, Em: ET485/30M
D(+)GlucoseSigma-AldrichG7021
Eclipse Ti-E MicroscopeNikonDiscontinued
Eppendorf epTIPS Boxes and Refill Trays, Volume: 0.1 to 10 µL, Length: 3.4 cm, 1.33 in., PP (Polypropylene)Eppendorf North America Biotools22491504
Eppendorf epTIPS Boxes and Refill Trays, Volume: 50 to 1000 µL, Length: 7.1 cm, 2.79 in., PP (Polypropylene)Eppendorf North America Biotools22491555
Ferrous Sulfate Acs 500 gFisher Scientific706834
FijiFiji (imagej.net)
Fisher BioReagents LB Broth, Miller (Granulated)Fisher ScientificBP9723-2 
Glass Cover SlideFisher Scientific12-542B 
Kanamycin SulfateSigma-Aldrich1355006
Magnesium sulfate Cert AcFisher ScientificXXM63SP3KG
Microscope HeaterWorld Precision Instruments96810-1
Potassium Phosphate MonobasicFisher Scientific17001H
ProScan III StagePrior
Red Filter CubeChromaEx: ET560/40X, Em: ET645/75M
Sapphire 561 LP LaserCoherent1170412
Slide, MicroscopeFisher Scientific125535B
Thiamine HydrochlorideSigma-Aldrich (SIAL)T1270-100G
Ti-LU4 Laser LaunchNikon
Yellow Filter CubeChromaEx: Z514/10X, Em: ET535/30M

References

  1. Cowley, M., Oakey, R. J. Transposable elements re-wire and fine-tune the transcriptome. PLoS Genetics. 9 (1), 1003234 (2013).
  2. Schneider, D., Lenski, R. E. Dynamics of insertion sequence elements during experim....

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