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The Continuous Evolution In Vitro Technique

Carolina Díaz Arenas1,  Niles Lehman1

1Portland State University, Portland, Oregon

Publication Name: 
Current Protocols in Nucleic Acid Chemistry
Unit Number: 
UNIT 9.7
DOI: 
10.1002/0471142700.nc0907s40
Online Posting Date: 
March, 2010
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Niles Lehman

Abstract

In vitro experimentation techniques were developed in response to the necessity of exploring new molecular structures and functions and to better understand evolutionary phenomena that shape organismal and molecular populations. The advancement of these techniques has allowed further exploration of more complicated evolutionary dynamics. One such technique is the continuous evolution in vitro (CE) method, to which this unit is devoted. The CE method is characterized by continuous cycles of amplification of RNA molecules that occur without much participation of the researcher. This feature allows us to evolve lineages in which the evolutionary phenomena occurring at the molecular level more closely mimic what happens in organismal populations in the present, or what may have happened in RNA populations during the RNA world stage of life. Curr. Protoc. Nucleic Acid Chem. 40:9.7.1-9.7.16. © 2010 by John Wiley & Sons, Inc.

Keywords: evolution; in vitro; mutation; RNA

     
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Table of Contents

  • Introduction
  • Basic Protocol: Continuous Evolution In Vitro (CE) Experiments
  • Alternate Protocol: Continuous Evolution In Vitro (CE) Experiments Using a Microfluidics System
  • Support Protocol 1: Determining the Dilution and Amplification Scheme
  • Support Protocol 2: Preparation of Ligase Ribozymes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Topics
    • Nucleic Acid Chemistry
    • Molecular Biology
     
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Materials

Basic Protocol: Continuous Evolution In Vitro (CE) Experiments

 Materials
  • Stock ligase ribozymes [50 µM] (prepared in advance; see Support Protocol 2)
  • CE buffer (prepared in advance; see recipe)
  • NTP mix (see recipe)
  • DEPC-treated and/or RNase-free water
  • PE mix (see recipe)
  • Ice bucket
  • 600-µL and 1.5-mL tubes
  • Timers
  • Benchtop cooler
  • 37°C heating block
  • Quick-spin minifuge (ISC BioExpress, cat. no. C1301)
  • Additional reagents and equipment for carrying out PCR (e.g., Kramer and Coen, 2001) and agarose gel electrophoresis (e.g., Voytas, 2000)

Support Protocol 1: Determining the Dilution and Amplification Scheme

 Additional Materials (also see Basic Protocol)
  • -32P-ATP (10 µCi/µL)
  • 2× XC– (bromphenol blue with no xylene cylenol added) acrylamide gel-loading dye
  • 5% polyacrylamide/8 M urea gel
  • Phosphorimager
  • Additional reagents and equipment for carrying out denaturing PAGE (e.g., see appendix 3A)
Looking for materials?  Suppliers Guide
     
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Figures

  • Figure 9.7.1
    The CE system. The continuous in vitro evolution (CE) scheme showing the reaction cycles where the ribozymes are amplified. All reaction steps take place in one homogeneous environment. The cycles start when the ribozymes (secondary structures shown at the top) catalyze the ligation of the trans oligomer substrate to themselves. Next, a reverse transcription amplification occurs and the cDNA copies are then transcribed if they posses the T7 RNA polymerase promoter sequence, which is contained in the substrate. At this point, cDNA products of nonreacted ligases are not transcribed, while cDNA copies with the (+) prom sequence are transcribed to initiate another cycle. RNA strands are represented by thick solid lines, while the cDNA strands are represented by thin solid lines.

    View Image
  • Figure 9.7.2
    Photograph of the CE hood that shows its organization inside and its surroundings. Next to the hood, at the right side, notice the ice bucket, the small bench cooler where the PE mix is kept, and the small freezer dedicated to the CE experiments. Inside the hood, there are two distinctive areas with a spatial separation between them: the reaction area and the dilution area. The reaction area is localized towards the right side of the hood. It is constituted by the timers, the 600-µL rack that has attached to it five holders for 1.5-µL tubes (for CE buffer and three dilution tubes), the heat block, pipet tips, and a dedicated pipet for the reactions. Notice that this area is close to the side of the hood where the benchtop cooler is kept, to minimize spatial motion. Stock tubes with NTP mix and CE buffer are placed in this rack towards the right side, and kept closed at all times except when being in used. The 600-µL reaction tubes are kept in the rack by the far left side, and walked to the front when in use (e.g., 5 min before stopping the reaction). The dilution area is localized towards the left side of the hood and it is constituted by a water bottle, a couple of racks for 1.5-µL tubes, a pipet tip box, and a pipet dedicated to fill the dilution tubes, which are kept in these racks with their lids closed at all times. The dilution tubes necessary to stop a reaction are moved from these racks to the holders attached to the reaction rack.

    View Image
  • Figure 9.7.3
    Agarose gel analysis of population persistence or extinction. Agarose gel images of two populations evolved with the CE system. Numbers beneath the gels track the specific bursts within a CE lineage. The symbol (–) denotes a negative control in the PCR. Panel A depicts a healthy population, with the presence of a strong PCR band being sustained through time. Panel B depicts a population that is going extinct, with the disappearance of the proper sized band over time, a consequence of a lack of sufficient RNA amplification of the population during CE.

    View Image
  • Figure 9.7.4
    Amplification dilution scheme. During the CE, the ribozymes are subjected through cycles of amplification (bold lines). At the end of each cycle (burst), the amplified population size is subject to a dilution (dash lines) that serves two purposes: (1) stop the reaction, and (2) keep the effective population size (Ne) nearly constant on average. The x-axis indicates time and the y-axis indicates population size. Panel A shows a case in which the dilution matches the amplification and the Ne stays nearly constant. Panel B shows a case of underdilution and the Ne increases with time. Panel C shows a case of overdilution and Ne decreases with time; notice that by the end of the fifth burst the population size has dropped below zero (extinction). The cases shown in panels B and C are to be avoided because they mask increases or decreases of the Ne due to evolutionary dynamics.

    View Image
  • Figure 9.7.5
    Adjusting the amplification factor to maintain Ne. The number of minutes in each CE burst can be adjusted to achieve an RNA amplification that matches the dilution factor between bursts. These gels depict a population in which this match is correct. Panel A shows the PCR products resulting from the cDNA amplification of a healthy lineage as in Figure 9.7.3. Panel B shows a PAGE image of the same lineage in which -32P-ATP has been included in the CE mixture. The left-most lane is a positive control where reacted (R) and unreacted (U) class I ligase ribozymes could be located. Note that no RNA can be seen in any of the bursts, signifying that the RNA population is not being overamplified with time.

    View Image
  • Figure 9.7.6
    Burst length vs. Ne scheme. The length of the burst is given by the amount of amplification that can be achieved before nutrients become exhausted and side product buildup. The amount of amplification achieved given the nutrients provided is dependent on the population size. Therefore, the burst length is directly related to population size, as show. The x-axis indicates the burst length time (in minutes) and the y-axis indicated the population size. Approximate burst lengths and population sizes are given as an example.

    View Image
  • Figure 9.7.7
    B16-19 ligase ribozyme secondary structure. Secondary structure of the B16-19 ribozyme is shown, with the exogenous substrate. The ribozyme effects the ligation of this substrate oligomer (in gray) to itself by catalyzing the attack of the 3¢-hydroxyl group of the substrate onto the 5¢--phosphate of the ribozyme (arrow). The substrate contains the T7 RNA transcriptase promoter sequence that is required for the CE cycles to occur. The primer-binding sites for reverse transcription (3¢ end) and PCR amplification at the 5¢ end of the promoter are denoted by solid rectangles.

    View Image

Literature Cited

Literature Cited
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Author Notes

Niles Lehman
April 13, 2010

The reference Diaz Arenas & Lehman (2009b) has now been published. The formal reference for this paper is:

Díaz Arenas C, Lehman N (2010). Quasispecies-like behavior observed in catalytic RNA populations evolving in a test tube. BMC Evolutionary Biology 10:80

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Niles Lehman
Posted on April 2, 2010 - 3:26pm

The reference Diaz Arenas & Lehman (2009b) has now been published. The formal reference for this paper is:

Díaz Arenas C, Lehman N (2010). Quasispecies-like behavior observed in catalytic RNA populations evolving in a test tube. BMC Evolutionary Biology 10:80.

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