CRISPR Project Lab

CRISPRing lepidopterans to study wings

Fluorescence Project S18

Resources:

Readings:

Course Description:

Honors Genetics Lab is project-based, where students will design and conduct a laboratory experiment aimed at exploring aspects of molecular genetics using the fluorescent protein mutagenesis project. 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 conducting original research, 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.

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

  • Generate genetics hypotheses using a genetics model system.
  • 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.

Lab Schedule

(Subject to change, including assignment due dates)

DUE DATES

  • Final Lab Report (Required) – due Tuesday 4/24 to t2/Assignments by 11:55 pm
  • Final Lab Notebook (Required) – due electronically on bio2355.biosci.gatech.edu on Thursday 4/26 by 11:55 pm
  • Grant Proposal (Optional) – due Tuesday 5/1 to t2/Assignments by 11:55 pm.

OFFICE HOURS

  • Dr. Spencer: Mondays 10am-noon
  • Jenni: Wednesdays 2-4 pm
  • Carl: Tuesday 2-4 pm

SCHEDULE

Week 13 ( 4/12) – Writing Workshop

Before lab,

  • Download your fluorescence assay results (below) and interpret them. If time, incorporate them into your results for this week’s assignment.
  • Complete your Results section and Figures&Tables section (upload to t2 and bring print copy to class)
  • Complete the Pre-Lab 13 on t2/Pre-Labs.

In lab,

  • Interpreting results in light of your research question.
    • Here’s a video on fluorescence excitation wavelengths and emissions spectra. You’ll need to be logged into GT to view.
  • Writing Workshop: Writing a Discussion Section. Revising a draft.
  • Overview of Grant Proposal (2355 Grant Proposal Guidelines)

After lab,

  • Writing Assignment: Discussion section, two copies in lab and upload to t2/Assignments.
  • We will meet in Week 14 and have a pre-lab due before class.

Week 12 (4/5) – Fluorescence assay & results interpretation

In lab,

  • Quantify fluorescence for each ancestor and derived mutant genotype
  • For the Fluorescence Assay protocol, the prep lab staff completed these steps:
    • Tues PM, picked single colonies from last week’s re-streaked plates. Transferred to 5 ml LB+CARB (no iptg) and grew overnight at 37C
    • Wed, transferred 300 ul of each culture to 10 ml LB+CARB and grew to OD0.8 at 37C, about 2 hrs
    • Wed, added IPTG to a0.2mM final concentration and shook culture at room temp for 16 hrs
  • For Fluorescence Assay in lab this week:
    1. Aseptically transfer 1 ml of well-mixed culture to a microfuge tube and centrifuge at 14,000 rpm for 5 min
    2. Carefully pipet off and dispose of the supernatant without disturbing the cell pellet
    3. Resuspend the cell pellet in 200 ul fresh LB+CARB medium
    4. Follow TA directions when loading samples into 96 well plates
  • Peer review & working session for results section

After lab,

  • Update your lab notebook by Friday.
  • Download your fluorescence assay data, below, and analyze to incorporate into your results. We will also discuss these in class on Thursday, but you’ll want to have looked at them before you take the prelab.
  • Fluorescence Assay results: In the results below, the excitation wavelength was set to approximate the ancestral excitation, and lowered if the emission spectrum peaked near the excitation wavelength. If the sample fluoresced when excited, then the numbers (excel file) and data plots (png file) show the emission spectrum (the colour at which the sample fluoresces) for that sample.  All sample data were blanked before plotting, so the usable data are in the table called “Blank EM Spectrum.” Because the excitation wavelength and emission spectra overlap, the emission data show the external excitation spike, labeled as “overflow.” Overflow and surrounding high intensity values should be omitted from analysis.
  •  Determine the peak emission wavelength for your samples using the files below:
  • Writing Assignment: Results section due in lab (1 copy) and on t2. (If you have an extension for the draft, you’ll have a tight turnaround on any email feedback; consider a visit to office hours.)

Week 11 (3/29) – Sequencing Analysis & Fluorescence quantification preparation

In lab,

After lab,

  • Complete the analyses you need to address your research question.
  • Update your lab notebook by Friday.
  • Writing Assignment: Draft your Results section using the RUBRIC and guidelines. Upload a copy to t2 and bring TWO print copies to class.

 

Week 10 (3/15) – Gel Electrophoresis & Sequencing

In lab,

  • Gel Electrophoresis to confirm PCR amplified the gene of interest
  • Discussion: What results will sequencing generate and what would you like to do with those results?
  • Determine which PCR products to prepare for sequencing. Prepare those products.

After lab,

  • Update your lab notebook by Friday. Your update should include your ideas for what direction you’d like to take the data analysis, and possibly what future directions this project could have (useful prep for writing a grant proposal based on our preliminary results).

 

Week 9 (3/8) – Select target colonies containing new fluorescent mutants & Colony PCR

In lab,

  • Assess transformants for fluorescence and makes notes on your product.
    • What makes a good clone?
  • At this point the dataset becomes a class project dataset. Make a class plan for every group to PCR and streak 5 mutagenized colonies (groups have first pick of their own plates).
  • Set up colony PCR on mutants and PCR the ancestral plasmids (for later sequencing)
  • Create a streak plate for each of your colonies for subsequent fluorescence analysis and characterization. Prep lab will create streak plates for the ancestral strains from their frozen stock.

After lab,

  • Update your lab notebook by Friday.

Week 8 (3/1) – Troubleshooting Transformations

In lab,

After lab,

  • Update your lab notebook by Friday.
  • Writing Assignment: Revise your Methods section using the RUBRIC, guidelines, and comments from the TAs.

Week 7 (2/22) – Select target colonies containing new fluorescent mutants

In lab,

  • Assay and photograph colonies under UV then streak target colonies
  • Lab Group Meeting – what’s happened to date, and what are the next steps?

After lab,

  • Update your lab notebook by Friday.
  • DUE next week in lab: Methods section, revised with feedback from last time, with all methodology to date and a bullet point outline of what you think needs to happen next. Bring one print copy to lab and upload to t2/Assignments.

Week 6 (2/15) – Transformations and plate cells

In lab,

  • Transform bacteria with your plasmid bearing the gene of interest, then and plate cells to screen for fluorescent gene expression.

After lab,

  • Update your lab notebook by Friday.

Week 5 (2/8) – Gel Electrophoresis & Ligation

In lab,

  • Your lab team will need to split into two groups to complete today’s bench work. Before you begin, make sure that each sub-team has a written out procedure that the entire team has agreed to, and get your TA to sign off.
    • Sub-team 1: Gel Electrophoresis of PCR product, undigested and digested plasmids
    • Sub-team 2: PCR clean up of digests, quantify DNA, and set up ligation

After lab,

  • Update your lab notebook by Friday.
  • DUE next week in lab: Methods section with all methodology to date and a bullet point outline of what you think needs to happen next. Bring two print copies to lab and upload to t2/Assignments.

Week 4 (2/1) – Digest (Wet lab)

In lab,

  • Clean up the PCR product, quantify DNA, and set-up a double enzymatic digestion set up
  • Each lab team partners with another team to review and explain each step from Weeks 2 and 3 at the whiteboard.
  • Peer-review of Introduction

After lab,

  • Update your lab notebook by Friday.
  • DUE next week in lab: Introduction section with at least three paragraphs and at least five citations. Bring two print copies to lab and upload to t2/Assignments.

 

Week 3 (1/25) – Journal Club & Plasmid DNA extraction (Wet lab)

In lab,

  • Plasmid DNA extraction, DNA quantification, and Mutagenic PCR
  • Journal Club on methodology and protocols

After lab,

  • Update your lab notebook with comments on the project plan and relevant background information from the article (using citations!).

 

Week 2 (1/18) – Journal Club & Project Planning (Wet lab)

In Lab,

  • Micropipette exercise and worksheet
  • Journal Club
  • Project Planning

After lab,

  • Update your lab notebook with comments on the project plan and relevant background information from the article (using citations!).
  • download and read  Shaner et al 2004 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.

Week 1 (1/11) – Project Overview (Dry lab)

Before Lab,

  • Please download and print a copy of Lippincott-Schwartz & Patterson 2003 (updated reading!), read the abstract, and make a list of at least three questions you would like clarification on.

In Lab,

  • Lab Safety talk and contract
  • review the syllabus and the plan for the semester
  • dissect a scientific paper for practice in advance of your reading assignment for next week

After lab,

  • Read Lippincott-Schwartz & Patterson 2003 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 online lab notebook on bio2355.biosci.gatech.edu/notebook, following these instructions on the course website.

 

BELOW IS TENTATIVE SCHEDULE INFO FOR REMAINDER OF SEMESTER, SUBJECT TO CHANGE

           

Week 10 (3/15) – Analyze results

In lab,

  • Analyze individual group PCR results – 1 hr
  • Writing exercise: Draft your results section. Run you ideas by your peers and TAs.

After lab,

  • Update your lab notebook by Friday.
  • DUE next time in lab: Results section with images and figures, and a bullet point outline of points for your discussion. Bring two print copies to lab and upload to t2/Assignments.

 

Week 11 (3/22) – SPRING BREAK

 

Week 12 (3/29) – Phylogenetic analysis

In lab,

  • Phylogenetic analysis of whole class sequencing data

After lab,

  • Update your lab notebook by Friday.

Week 13 (4/5) – TBD

In lab,

  • Writing workshop

After lab,

  • DUE: Final Lab Report. DUE in lab next week. Bring two print copies to lab and upload to t2/Assignments.

 

Week 14 (4/12) – TBD

In lab,

  • Grant proposal writing workshop

After lab,

  • Update your lab notebook by Friday.
  • Grant Proposal. DUE in lab next week. Bring two print copies to lab and upload to t2/Assignments.

Week 15 (4/19) – TBD

In lab,

  • Lab notebook workshop

After lab,

  • DUE: Lab notebook. DUE 4/26 by midnight