This week’s BEACON Researchers at Work post is by University of Texas postdoc Gwen Stovall.
The Student Connection
So, maybe the students didn’t have a good idea of “evolution” in the beginning. As far as they knew, they were unable to observe evolution… that takes eons to occur. New experiences, though, offered new insights and the students didn’t retain these misperceptions for long.
In the early stages of developing an “evolution kit” for use as a high school demonstration, undergraduate students from the Freshman Research Initiative (FRI) were recruited to design, build, and implement the system. The FRI is a novel program comprised of more than 40% underrepresented groups and provides authentic research experiences to students. It also provided a partnership for the educational outreach missions of this project.
Both undergraduate and high-school students have been instrumental in all aspects of this project. From its first inception to the later stages, many high school and undergraduate students have contributed to these efforts, offering their unique perspective. Ultimately, a single undergraduate, Michael Ledbetter, stepped up to lead the team. Much of the success of this project is attributed to his steadfast, “heads down” approach, which culminated with conference speaking engagements and a recent publication Biochemistry and Molecular Biology Education (Ledbetter et al., 2013).
The Science Driver (Overview)
Underlying the kit’s development, we have merged previously developed technologies by combining continuous selection methods with a colorimetric reporter. We recreated one of Dr. Joyce’s continuous evolution systems using a modified variant of the Bartel self-ligating ribozyme (Wright and Joyce, 1997; Bartel and Szostak, 1993). The selection begins with a pool of T500 ligase ribozymes randomized at all 3 nucleobases composing the catalytic core (“T500 N3” pool). Serial dilutions cause selective pressure driving the evolution of species with improved catalytic function. With only 3 mutations at essential positions, improved catalytic function is evident after only one round of selection, thus suitable for the timeframe of a classroom period. This continuous evolution scheme is paired with a fluorescent reporter assay (strand displacement based). Using a fluorescent signal minimizes the equipment required to observe the selection. Therefore, the selection for ribozyme ligation and subsequent amplification is observed via simple colorimetric signal. This technological feat lends itself well to high school labs which lack more complex molecular biology equipment.
The Return (More specifically)
So, what are the students actually doing? They are observing the selection of a ribozyme. A ribozyme is a strand of RNA with catalytic function. In this case, the ribozyme can attach (or ligate) another RNA strand to its end (5’ end). This addition (containing a T7 promoter) permits the enzymatic amplification of the ligating strand. Thus, only the RNA variants with ligase activity are amplified. Simple dilution of the pool (with the reaction reagents) and a single round of amplification increases the concentration of “winning” ribozyme(s). Just 1-3 rounds of selection results with a fairly decent ribozyme.
What do the students actually see? Upon performing 1-3 rounds of ribozyme selection, the students observe the success of the reaction using a fluorescent reporter assay. Once the RNA strand (containing the T7 promoter) is ligated to the ribozyme, the end of the ribozyme displaces the paired fluorophore-quencher oligonucleotides, thus generating a fluorescent signal upon excitement with a UV light. Specifically, using the assay, the students test each round of their selection for ribozyme (or ligase) activity, which results with an increase in fluorescence over the course of the rounds.
What do the students actually get out of the experience? In addition to exposure to many of the techniques and theories of molecular biology, the students receive a working knowledge of catalytic RNA, which provides some insight into the RNA world hypothesis. Additionally, as is the focus of the demonstration, the students receive a first-hand demonstration of a selection for function, a necessary component of evolution.
With the development of the first prototype of the “evolution kit” demonstration, we’re now focusing on testing, evaluation preparation, fine tuning the protocol, and getting it into the hands of high school students.
Shruti Singh, a former FRI student and current undergraduate mentor in the class, has now taken over the reins from Michael Ledbetter. Shruti, who recently presented the project at an undergraduate research conference at Rice University, is seeking to begin field-testing the prototypes with high school students soon. This will assess the feasibility of the required laboratory techniques for most high school students and if the tenets of this demonstration align with the educational curriculum. Most importantly, this will assess this experiment’s ability to introduce and reinforce fundamental evolutionary theory to high school students.
For more information about Gwen’s work, you can contact her at gwenstovall at gmail dot com.