CRISPR Project Lab

CRISPRing lepidopterans to study wings

Bee Project F18

Course Description:

This course is designed for exceptional students interested in learning important concepts and practical techniques in the field of genetics. Honors Genetics Lab is project-based, where students will design and conduct a laboratory experiment aimed at exploring aspects of transmission genetics, population genetics, and molecular genetics using the Georgia Tech Urban Honey Bee Project and neighboring hives. As with all research, we will begin with a question and then follow the scientific method to generate a hypothesis, design and conduct an experiment, and analyze the data to draw a conclusion. Because we’ll be exploring new questions to Atlanta’s urban honey bee populations, we’ll probably also bump into the primary frustrations of scientific research—assays that require troubleshooting, delays when protocols don’t work perfectly at first pass, and results that don’t match our thinking about the system. We’ll do this because asking real questions in a relevant study system is what scientists do, and learning how to navigate the process and solve the ensuing problems is the best training you can have  for your senior research experience and to pursue careers in scientific research, medicine & human health, or other fields that require problem solving and logic.

Course Goals: By the end of this course, you will be able to:

  • Generate and craft a thorough, genetics-based hypotheses about European Honey Bees.
  • Design experiments and interpret results using basic statistical analysis.
  • Create and troubleshoot genetics lab protocols.
  • Cite relevant genetics primary literature.
  • Write effective and accurate notebook entries, and lab reports in the style accepted by genetics scientific journals.
  • Use appropriate lab safety standards and precautions.

Due Dates

Weekly Schedule (link to new SCHEDULE)

11/15 – Week 13 – Results & Discussion

11/8 – Week 12 – Figures & Results

Important statistical reminders, in addition to what we covered in class:

11/1 – Week 11 – Data Analysis

Before lab,

  • Upload and bring print copy to lab of Writing Assignment: Introduction
  • Complete the prelab, which has no required reading but steps you through installing R on your laptop.
  • BRING COMPUTERS TO LAB!

In lab

After lab,

  • By midnight Saturday:
    • Update lab notebook. This is the last real update you’ll do on your lab notebook, as next week will be a writing and synthesizing workshop. Your entry should include what statistical approaches you’ll do, and how to do them, including what lines of code, links to data files, etc. You are not constrained to use R, but I wanted to expose you to this very useful and flexible programming language. Here’s a compelling blog post on why R is a valuable data science tool:
      https://www.r-bloggers.com/why-you-should-learn-r-first-for-data-science/
    • Proofread  the class dataset.
  • Due in lab next week:
    • Writing 6 – Dataset & preliminary analysis: Create your own data set file for analysis in R, and then run the appropriate analysis for the way you’ve structured your data. Upload the dataset and the output of your analysis here. Note that you are not obligated to use R if you have access to and facility with another stats package.
  • No Prelab. Good luck on the lecture exam!

10/25 – WEEK 10 – Gels and data assembly

Before lab,

  • Writing Assignment: Write or revise your Methods paragraph on your urbanization metric (what is it and how with you measure it–with citations). Upload to Canvas and bring a print copy to class.
  • Recall that our hive locations file is linked on Canvas in the Announcements–and also in your email from 10/4/18. Please do not share the details of these hive locations with anyone outside our class. Lab Notebooks incorporating this link should have visibility set to private.
  • Complete the Prelab on Canvas (opens Monday).
  • BRING LAPTOPS TO LAB!

In lab,

  • Run your 12 PCR products in the gels. Please arrange them as follows for ease of interpretation. loading 10 ul in:
    •  For each sample, combine your five loci into 2 wells, carefully recording what is in each well. Keep the negative control in its own well. Electrophorese your PCR products on a 2.0% agarose gel at 100 V for 60-80 minutes, or until good size separation for PCR products ranging from 150–775 bp. Record the gel number and gel lanes that contain your samples. Visualize using the gel doc, calculate band sizes against the known sizes and the DNA marker, print your gel (invert the image—bands dark, gel background light) for your lab notebook and lab report, and record your results.
  • Before you leave lab, add your sample results for presence/absence of each RNA virus to the class dataset gsheet shared by Canvas announcement.
  • Working Session – Urbanization metrics
  • Introduction Guidelines
  • Gel results (emailed to you and posted after lab)

After lab,

  • Update your lab notebook and add your data to the class gsheet (we’ll do the gsheet data pooling in lab next week).
  • Finalize your urbanization metric and categorize each colony.
  • Complete the Prelab by Wednesday midnight.
  • Writing Assignment: Introduction, written using the Lab Report rubric and by referencing the Introduction sections of the other articles we have read as a model. Remember though that every word you write should be from your own thoughts rather than modification of the works and words of others. We will grade from the rubric sections: Introduction, Literature Cited, Writing, Format. Upload to Canvas/Assignments and bring a print copy to lab.

10/18 – WEEK 9 – PCR on cDNA 

Before lab,

  • No prelab this week.

In lab,

  • PCR on all five cDNA samples with five RNA virus loci
  • Characterizing urban and rural
    • Literature searches
    • Databases
    • Online software
    • The link to our data can be found on the course Canvas site (behind the login firewall)
  • Workshop Introduction

After lab,

  • Update your lab notebook
  • Writing Assignment: Introduction uploaded to Canvas as well as bringing a print copy to class.
  • Complete the PreLab.

10/11 – WEEK 8 – RNA isolation

In lab,

After lab,

  • Update your lab notebook by Friday midnight.
  • Complete the TA evaluation and the Course Midterm Evaluation on Qualtrics by Sunday at midnight. (Check your email for a message from “noreply@qemailserver.com.”
  • Revise your methods and upload to Canvas/Assignments by Sunday at midnight.
  • No pre-lab for next week

10/4 – WEEK 7 – Methods & Gel Interpretation

Before lab,

  • Writing Assignment: Write your METHODS section for our experiment. Use the Lab Report rubric and reference the Methods sections of the other articles we have read as a model. Remember though that every word you write should be from your own thoughts rather than modification of the works and words of others. Upload to Canvas/Assignments/ “Writing 2 – Methods” and bring a print copy to lab.
  • Complete the Prelab on Canvas.

In lab,

  • Run PCR samples in 2% agarose at 100 V for 45-60 minutes.
  • Methods writing workshop while gels are running.
  • Size the bands on your gel and record the results.
  • Peer-review of methods after gels are done
  • Results:

After lab,

  • Update your lab notebook.
  • Complete the TA evaluation and the Course Midterm Evaluation on Qualtrics by next Friday (10/12) at midnight.
  • No writing assignment or prelab. Happy Fall Break and good luck on the lecture exam!

9/27 – WEEK 6 – cDNA PCR / Multiplex pilot

Before lab,

  • Quickly read through the PCR protocol and then complete the PreLab. This week’s PreLab is open-ended questions to get some feedback from you.

In lab,

After lab,

  • Update your lab notebook
  • Writing Assignment: Methods section for the full project, omitting the statistical analysis but including everything else. Use the rubric to guide your writing.

9/20 – WEEK 5 – RNA isolation

Before lab,

  • Update lab notebook by Friday, including a revision of your hypothesis using feedback from lab, using the Lab Notebook Rubric. 
  • Consider, are there bee genes with known primers that you’d like to explore? Next, review the protocol we’ll use to isolate RNA and synthesize cDNA, based on the kit instructions you looked at in class. Finally, complete the PreLab before Wednesday midnight.
  • Here’s a brief overview of the counts of samples that contained each of five bee viruses. There were 20 hives total, and each hive was sampled 5 times:
    • Sample DWV (B) SBV (C) IAPV (A) BQCV (D) CBPV (E)
      Hives with virus 13 7 4 2 0
      Samples with virus 24 10 8 2 0

In lab,

After lab,

  • Update your lab notebook
  • Writing Assignment (TBD)

9/13 – WEEK 4 – Gel Electrophoresis

Before lab

  • Read the lab handout on Agarose Gel Electrophoresis. Complete the PreLab before Wednesday midnight.
  • Writing Assignment: State our group research question and from it create a detailed hypothesis in your own words. List three background pieces of evidence that you’ll need to support your hypothesis; one of these should include an idea and citation for how to define urban v. rural. The remainder of ideas do not need to be supported with citations at this point. Submit to Canvas/Assignments before class on Thursday 9/13.
  • Journal Genetics Citation Format

In lab,

  • Practice loading a water sample into a gel with loading dye.
  • Example your PCR tubes–do they both have the same volume of liquid? Makes notes for you PCR protocol if your reactions weren’t 25 ul. Load 10 ul of your PCR product. and run gel of PCR products for ~1 hour at 100 V. While the gels are running we’ll work on hypotheses and protocols.
DNA Marker for Electrophoresis

Gels:

  • Hypothesis Score Card
  • How to create a protocol
  • Image your gel. Analyze the outcomes. After class, your TAs will post the gels to the website by group #.
    • Did your PCR work? Was it contaminated? How do you know?
    • If it didn’t work, what are some possible reasons? What information do you need to determine the cause of the failure? Discuss with your benchmates and a TA.

After lab,

  • Update lab notebook by Friday, including a revision of your hypothesis using feedback from lab, using the Lab Notebook Rubric. 
  • Complete the PreLab Week 5 due Wednesday by midnight.
  • Writing Assignment TBD

9/6 – WEEK 3 – PCR Pilot

Before lab

In lab,

  • This week we put your pipettor and quantitative skills to the test.
  • PCR pilot of bee cDNA using primers for 5 different viruses (Sguazza et al. 2013)
    • PCR pilot protocol
    • PCR Template = copy DNA from Apis mellifera samples collected in 2016
    • PCR profile: 30 cycles of
        • 94 °C for 30 sec
        • 56 °C for 30 sec
        • 72 °C for 45 sec
  • Discussion of Sguazza et al 2013 detection of bee viruses by multiplex pcr
  • Finalize a project question and plan our experimental design.
  • Conducting a literature search.

After lab,

  • Watch the video on PCR (this is proprietary library resource, so you’ll need to log in to the library or view from a campus computer) to gain a more nuanced understanding of PCR
  • Writing Assignment: State our group research question and from it create a detailed hypothesis in your own words. List three background pieces of evidence that you’ll need to support your hypothesis; one of these should include an idea and citation for how to define urban v. rural. The remainder of ideas do not need to be supported with citations at this point. Submit to Canvas/Assignments before class on Thursday 9/13.

8/30 – Week 2 Benchwork Intro and Journal Club (meet in CE 123)

Before lab,

  • Download and read Youngsteadt et al 2015 using the basics provided in “dissecting a scientific paper.” Bring a copy of the paper and your reading notes to class next time to help facilitate discussion.
  • Create your lab notebook and title it: F16A or B – Last, First Name – Project Name. For now you can leave the project name undecided, and update that once we’ve settled on a research question (due by Friday 8/26).
  • Complete the PreLab Week 2 on Canvas/Quizzes before Wednesday midnight.
  • Come dressed for wet lab: lab coat, long pants, close-toed shoes.

In lab,

  • Lab safety, Safety quiz and paperwork
  • Pipettor introduction and lab skills
  • Discussion of Youngsteadt et al 2015 
  • Project planning
  • Make your video

After lab,

  • Update your lab notebook with your own, solo-written research question that meets the benchmarks we discussed in lab. It may be the same or similar to one of those presented in class or completely different. Regardless, it should be your original work in your own words.
  • Complete your Pre-lab Week 3 by Wednesday midnight. (Opens on Monday.)

8/23 – WEEK 1 – Bee Overview (meet in CULC 481 this week only!)

Before lab

  • Please read: BeeBasicsBook before lab (read pages 1-12, 30, 31)

In lab,

  • We’ll solicit student learning goals for the course, learn basic bee biology & tour the beehives on campus.
  • We’ll meet our off-campus collaborators virtually and learn what types of hive samples they can donate to our research project.
  • After we review the syllabus and the plan for the semester, we’ll consider How to read a scientific paper for practice in advance of your reading assignment for next week.

After lab,

  • Download and read Youngsteadt et al 2015 using the basics provided in “How to read a scientific paper.” Bring a copy of the paper and your reading notes to class next time to help facilitate discussion.
  • Create your lab notebook and title it:
    • F18A or F18B – Last, First Name – Lab Notebook

Guidelines and Rubrics:

Protocols

Readings

Urban-Rural Articles and Citations

  • McIntyre NE, Knowles-Yánez K, Hope D. 2008. Urban Ecology as an Interdisciplinary Field: Differences in the use of “Urban” Between the Social and Natural Sciences. Pages 49-65 in Marzluff JM, Shulenberger E, Endlicher W, Alberti M, Bradley G, Ryan C, Simon U, ZumBrunnen C, eds. Urban Ecology: An International Perspective on the Interaction Between Humans and Nature. Boston, MA: Springer US.
  • Medley, K. E., McDonnell, M. J. and Pickett, S. T. A. (1995), Forest-Landscape Structure along an Urban-To-Rural Gradient*. The Professional Geographer, 47: 159–168. doi:10.1111/j.0033-0124.1995.00159. http://onlinelibrary.wiley.com/doi/10.1111/j.0033-0124.1995.00159.x/abstract
  • Hall, S. A., J. S. Kaufman, and T. C. Ricketts. 2006. Defining Urban and Rural Areas in U.S. Epidemiologic Studies. Journal of Urban Health 83:162-175.
  • Branch, G. P. (2015, July 27). Urban and Rural Classification. Retrieved September 14, 2016, from https://www.census.gov/geo/reference/urban-rural.html
  • “2010 CENSUS – URBANIZED AREA REFERENCE MAP: Atlanta, GA 34.590397N.” ICPSR Data Holdings (2010): n. pag. United States Census Bureau. US Department of Commerce, 22 May 2012. Web. 12 Sept. 2016.
  • Shugarman LRP, Sorbero MESP, Tian HP, Jain AKMS, Ashwood JSMA. 2008. An Exploration of Urban and Rural Differences in Lung Cancer Survival Among Medicare Beneficiaries. American Journal of Public Health 98:1280-1287.
  • Bates, A. J., Sadler, J. P., Fairbrass, A. J., Falk, S. J., Hale, J. D., & Matthews, T. J. (2011). Changing Bee and Hoverfly Pollinator Assemblages along an Urban-Rural Gradient. PLoS ONE, 6(8). doi:10.1371/journal.pone.0023459
  • Branch, Geographic Products. “2010 Census Urban Area Reference Maps.” US Census Bureau    Geography. U.S. Department of Commerce, n.d. Web. 15 Sept. 2016.
  • McDonnell, Mark J. “Ecosystem Processes along an Urban-to-rural Gradient.” Urban Ecosystems (1997): 21-36.

Background Articles