Genome to phenome: DNA-protein interactions involved in butterfly wing colored development

Michelle Borrero, University of Puerto Rico- Rio Piedras Campus

Anthony R Rivera Barreto

Jessica M Rodriguez Rios

Jose A. Rodriguez Martinez

Riccardo Papa

Location: Puerto Rico

Abstract

We are interested in understanding the genomic mechanisms underlying morphological differences within species. We will use the wing color pattern of Heliconius erato as a model. We have developed a course-based undergraduate research experience (CURE) that will engage undergraduate biology majors in the identification and purification of transcription factors in butterfly wing development. Through this experience students will be able to use the knowledge and concepts from the literature to make and defend decisions, explain the role of DNA binding proteins in the genome to phenome relationship and recognize the application and utility of the techniques used in the research for their career development.

Student Goals

  1. Use knowledge and concepts from literature to make and defend decisions
  2. Explain the role of DNA binding proteins in the relationship between genome to phenome.
  3. Recognize the application and utility of the techniques using research ideas or goals.

Research Goals

  1. Identify and purified transcription factors involved in butterfly wing color development.
  2. Characterized genome-wide interactions between transcription factor underlying wing color patter development and DNA.

Context

This is one of four mid-level (3000) stand-alone laboratories that are offered for Biology majors to choose two as a graduation requirement. The course has Genetics as a pre-requirement. The duration of the course is of one semester. It meets 3 hours/ week and is equivalent to 1 credit course for the students. A maximum of 5 sessions are offered per semester with a maximum enrollment of 16 students per session.

Target Audience:Major
CURE Duration:A full term

CURE Design

First weeks are for skill development:

  • Pipetting exercise to ensure similar skill trough the students.
  • Basic bioinformatic skills & software for primer design and design of cloning strategy

Throughout the semester:

  • Provide videos of the techniques to familiarize students with the procedures.
  • Pair students with research experience with those that don't have prior knowledge.
  • Provide computers that already carry the necessary software that is needed to achieve the goals, and the input/data to practice with them.
  • Virtual design of the activities/laboratories for a low risk-setting.

We will include an experimental design workshop in the course

Each laboratory session has 16 students. Students work in pairs and we will have two pairs of student working with towards the cloning of the same gene. The rationales is to have duplicates of each cloning effort. We will include a visit to the Sequencing and Genotyping Facility (SGF) at UPR when DNA samples will be loaded for sequencing.

At the end of the project students will do oral presentations on their accomplishments and will propose what will they do next with each of their clones.

Core Competencies:Analyzing and interpreting data, Asking questions (for science) and defining problems (for engineering), Planning and carrying out investigations
Nature of Research:Basic Research

Tasks that Align Student and Research Goals

Research Goals →
Student Goals ↓
Research Goal 1: Identify and purified transcription factors involved in butterfly wing color development.
Research Goal 2: Characterized genome-wide interactions between transcription factor underlying wing color patter development and DNA.


Student Goal 1: Use knowledge and concepts from literature to make and defend decisions

Use bioinformatics(BLAST) analysis to identify a gene of interest.
Download the target gene in multiple species and perform alignment analysis to determine sequence similarity.
Design primers to amplify the gene of interest.
Identify a plasmid for gene cloning and protein expression.
Select appropriate control for the experiment.
Diagram study design (flowchart)

Formulate a hypothesis and design an in silico experiment to test it.
Identify the DNA regulatory elements utilizing custom made bioinformatic genomic analyses.
Propose possible genes under the controlled of specific transcription factor by searching for nearby coding sequences (in case of cis-regulation).
Make written report.



Student Goal 2: Explain the role of DNA binding proteins in the relationship between genome to phenome.

Search in PubMed or Web Of Science the primary literature using a review as a starting point. Propose at least 5 examples of transcription factor genes and their function. Present a summary of the one paper Journal Club format. Make an oral presentation with the results of the project.

Search in PubMed or Web Of Science the primary literature using a review as a starting point.
Present 1 example of a well characterize gene regulatory genetic architecture. ( example: Describe what is known about the regulation of agouti in mice color fur.)
Make an oral presentation with the results of the in silico project.



Student Goal 3: Recognize the application and utility of the techniques using research ideas or goals.

Identify possible careers and a job/internship that the acquired knowledge will allow.
Generate a resume and a cover letter that includes the skills/techniques develop in the course.

Identify possible careers and a job/internship that the acquired knowledge will allow.
Generate a resume and a cover letter that includes the skills/techniques develop in the course.


Instructional Materials

Worksheet for primer design hoja de cotejo tarea de primers.docx (Microsoft Word 2007 (.docx) 1.4MB Jun26 19)

Worksheet for written report of cloned gene sequence analysis Asignacion Bioinformatica 2018.doc (Microsoft Word 30kB Jun26 19)

For PCR primer design, cloning procedure, sequence analysis:


Assessment

The following assessment instruments are in Spanish:

Rubric to evaluate electronic notebook rubrica de libreta electronica biotec 2018.doc (Microsoft Word 53kB Jun26 19)

Rubric to evaluate oral presentations Rubrica-Presentacion Propuesta CURE.pdf (Acrobat (PDF) 70kB Jun26 19)

The objective of experimental design was evaluated with the E-EDAT in pre/post format (Brownell, S.E., et al., 2014)

Resume evaluation form (https://www.westga.edu/student-services/careerservices/assets-careerservices/docs/resume-evaluation-form.pdf)


Instructional Staffing

Michelle Borrero instructor and laboratory coordinator.
Jose Rodriguez and Riccardo Papa are the research leads.
Anthony Rivera is the graduate students affiliated to research project.
Maria del Pilar Ortiz was the lead research undergraduate student.

We had additional undergraduate research students facilitating the preparation of reagents and supplies for the lab. These were: Eduardo Perez, Maria Anca, Leyshka Pena, Jose F. Rivera, and Aml Smaili. These students dedicated about 5 hours/week each (except for Eduardo Perez who dedicated 10 hours/ week) to aid in the preparation of the lab.

All personnel have been involved in the CURE design except for the undergraduate students. These were informed about their role in the CURE before implementation.

Author Experience

Michelle Borrero, University of Puerto Rico-Rio Piedras Campus

I have been involved in developing experiences in laboratory courses to encourage the development of scientific process skills in our undergraduate students. These skills (e.g., gathering and analyzing data, experimenting, communicating results, etc) are at the core of our Department's graduate profile. Thus, the significance of these laboratory courses in our curriculum. From its inception, the Molecular Biotechnology Laboratory course was designed as a project-based laboratory experience. Hence, evolving into a CURE was a natural fit. As this course is targeted to juniors and seniors, my goal is for them to experience the research process to develop the skills that will enable them to be successful in either graduate or professional (medical) school. It was very gratifying to observe how they quickly acquired ownership of their research project, and how they were committed to obtain mastery of the technical aspects of the project, as well as understanding their contribution to the research project as a whole. I am looking forward to continue the use of CUREs into other mid- and upper- level laboratory courses at UPR as I was able to experience first hand their impact and effectiveness in our students.

Advice for Implementation

Our biggest challenge in the implementation of the course was the coordination of preparing reagents (e.g., broth, LB plates, buffers, etc) to have them available for the students according to their progress in the CURE. I recruited undergraduate research assistants that helped me in that process, as I did not have a laboratory technician or graduate teaching assistant assigned to the course. We prepared aliquots of all the reagents that were needed throughout the semester. For us it was helpful to have an additional space were students could come in to repeat experiments or techniques that presented a challenge. For this project you need basic molecular biology reagents and equipment. We pre-order different sets of primers prior to the beginning of the project, as well as the different kits that are required for the cloning process. We are fortunate that there is a sequencing facility on Campus, directed by Dr. Papa, that was willing to process our students' samples for free and allowed us to visit their facilities.

My experience was that my original plan (see syllabus) was very ambitious. Students needed time to develop the skills of almost every technique that we did in the course, which resulted in approximately 2 weeks/ cloning step. the net result was that we were only able to clone and sequence our genes of interest.

End of course evaluations showed that students really enjoyed, and learned, a lot through the CURE. They felt it was an excellent experience, particularly for students that are not part of a research group or that have not had a previous research experience. Their main criticism was that they felt that it was 'a lot of work for 1 credit' and that we should add more structure to the laboratory notebook.

Iteration

cDNA from H. erato was not available for the first iteration of the course on August, 2018. Thus, we chose to clone homologous transcription factors from D. melanogaster. Specifically, we cloned Sinus oculis, Six4, and Optix. Primer design and PCR reaction conditions were optimized the summer prior to implementation. For course syllabus (Spanish), click here Silabo del Laboratorio de Biotecnologia - CURE 2018-19.pdf (Acrobat (PDF) 194kB Jun25 19). We did a pilot session (16 students) and we allowed two iterations for critical steps throughout the semester: PCRs, Gibson Assembly, and transformation. Although the course intended to clone the genes and express the protein for these transcription factors, due to the iteration process that students had to do we were only able to clone and sequence the genes prior to their end of semester's oral presentation. These clones were given to the researcher team for experimentation. For next semester (Fall 2019), we plan to generate a cDNA library from H. charithonia and clone transcription factors important for the wing color development of these butterflies.

Using CURE Data

Transcription factors that are effectively cloned and/or expressed will be forwarded to the researcher affiliated to this project for further characterization. For quality purposes the DNA clones obtain will be send for sequencing and analysis. The teaching team will be in close communication with the research team for updates on student research progress.

Resources

Brownell, S.E., Wenderoth, M.P., Theobald, R., Okoroafor, N., Koval, M., Freeman, S., Walcher-Chevillet, C.L., and A.J. Crowe. (2014) How Students Think about Experimental Design: Novel Conceptions Revealed by in-Class Activites. BioScience 64, 125-137.

About transcription factors:

Campbell ZT, Bhimsaria D, Valley CT, et al. Cooperativity in RNA-Protein Interactions: Global Analysis of RNA Binding Specificity, Cell Rep , 2012, vol. 1 (pg. 570-81)

Kojima, T., Kunitake, E., Ihara, K., Kobayashi, T., & Nakano, H. (2016). A Robust Analytical Pipeline for Genome-Wide Identification of the Genes Regulated by a Transcription Factor: Combinatorial Analysis Performed Using gSELEX-Seq and RNA-Seq. PloS one.

Reed RD et al. (2011). Optix drives the repeated convergent evolution of butterfly wing pattern mimicry. Science 333, 1137–1141. (doi:10.1126/science.1208227)

Stormo, G. D. & Zhao, Y. (2010) Determining the specificity of protein–DNA interactions. Nature Rev. Genet. 11, 751–760.


Zhang, L., Mazo-Vargas, A., & Reed, R. D. (2017). Single master regulatory gene coordinates the evolution and development of butterfly color and iridescence. Proceedings of the National Academy of Sciences of the United States of America, 114(40), 10707–10712. http://doi.org/10.1073/pnas.1709058114