Guidelines for maintaining your lab notebook (PDF)
Guidelines for working in the tissue culture facility (PDF)
There are six stations for you and your lab partner to visit during orientation. Some will be guided tours with a TA or faculty there to help you and others are self-guided, leaving you and your partner to try things on your own. Your visit to each station will last 10-15 minutes. It doesn't matter which station you visit first but you must visit them all before the end of orientation. Your lab practical next time will assess your mastery of each station.
Introduction to Pipetting (PDF)
Introduction to our Microscopes (PDF)
Introduction to our "Back Room" and Tissue Culture Facility (PDF)
Introduction to Making Solutions (PDF)
Introduction to our Spectrophotometer (PDF)
Introduction to Lab Math (PDF)
Orientation Assignment (PDF)
In this experimental module you will modify the gene for EGFP (Enhanced Green Fluorescent Protein) to truncate the protein it encodes. Cells expressing the full-length protein glow green when exposed to light of the appropriate wavelength. You will be designing and then creating an expression vector to delete the first 32 amino acids of EGFP. Cells transfected with your expression vector should not glow green, a prediction you will test. You will also test whether this N-terminally truncated EGFP can recombine with a C-terminally truncated version to regenerate full length EGFP in vivo. Finally, you will have the opportunity to suggest changes to the experimental protocol that will increase the frequency of green cells in which there has been an inter-plasmid recombination event. We will then choose a few variables to test on the final day of the experiment.
A Schematic Overview of the Module (JPG) (Image by Prof. Bevin Engelward)
Timetable of the Module (JPG)
| SES # | LABS | INTRODUCTIONS | PROTOCOLS | REAGENTS LISTS | ASSIGNMENTS |
|---|---|---|---|---|---|
| 2 | DNA Engineering by PCR | (PDF) | (PDF) | (PDF) | (PDF) |
| 3 | Clean and Cut DNA | (PDF) | (PDF) | (PDF) | (PDF) |
| 4 | Agarose Gel Electrophoresis | (PDF) | (PDF) | (PDF) | (PDF) |
| 5 | DNA Ligation and Bacterial Transformation | (PDF) | (PDF) | (PDF) | (PDF) |
| 6 | Examine Candidate Clones | (PDF) | (PDF) | (PDF) | (PDF) |
| 7 | Restriction Map, Article Discussion/Tissue Culture | (PDF) | (PDF) | (PDF) | (PDF) |
| 8 | Lipofection | (PDF) | (PDF) | (PDF) | |
| 9 | FACS Analysis | (PDF) | (PDF) | (PDF) |
In this experimental module you will study an enzyme with a remarkable history, beta-galatosidase. "Beta-gal" as it's affectionately called had a starring role in the development of the operon model for gene regulation and continues to be a lab workhorse for gene expression studies. In bacteria, this enzyme hydrolyzes the disaccharide, lactose, into two simpler sugars, glucose and galactose. You will be measuring the efficiency of the enzymatic reaction using an artificial substrate, ONPG, which yields a yellow product when it is cleaved by beta-gal. Using a specialized bacterial strain, you will overexpress beta-gal to purify and analyze. Finally, you will test the effect of replacing a natural amino acid in beta-gal with an unnatural amino acid of your choosing, looking at the effect of such a substitution on the expression and activity of the modified enzyme.
| SES # | LABS | INTRODUCTIONS | PROTOCOLS | REAGENT LISTS | ASSIGNMENTS |
|---|---|---|---|---|---|
| 10 | Tools for Protein Engineering | (PDF) | (PDF) | (PDF) | (PDF) |
| 11 | Assessing Beta-galactosidase | (PDF) | (PDF) | (PDF) | (PDF) |
| 12 | Purifying Beta-galactosidase | (PDF) | (PDF) | (PDF) | (PDF) |
| 13 | Student Presentations | (PDF) |
Nucleotides have been called the building blocks of life, but as you've seen it's not trivial to build something with them. Recall you spent nearly three weeks truncating the gene for GFP, and your efforts to rationally modify an enzyme yielded a complex mixture of proteins and results. If one reasonable definition of a biological engineer is someone who builds things from biological materials, then let's get building. We'll start with an existing and clever design that uses bacteria as the pixels in a photograph. The bacterial cells have been engineered to respond to light, churning out a familiar enzyme (beta-gal) to turn the media black in the dark. You'll be using this system to take black and white pictures, identifying experimental changes that can affect the operation of system. You will also invert the logic in the existing design and add a red-fluorescent protein in the readout to generate two-color pictures. Finally you'll use quantitative PCR to characterize the simple or complex circuit, cataloging your findings at the Registry of Standard Biological Parts for future biological engineers to "build" on.
| SES # | LABS | INTRODUCTIONS | PROTOCOLS | REAGENT LISTS | ASSIGNMENTS |
|---|---|---|---|---|---|
| 14 | Tools for Systems Engineering | (PDF) | (PDF) | (PDF) | |
| 15 | Basic Bacterial Photography: Black and White | (PDF) | (PDF) | (PDF) | |
| 16 | Advanced Bacterial Photography: 2 Color | (PDF) | (PDF) | (PDF) | |
| 17 | Device Characterization | (PDF) | (PDF) | (PDF) | (PDF) |
| 18 | Measuring DNA, RNA, Protein | (PDF) | (PDF) | (PDF) | (PDF) |
| 19 | RT-PCR Data Analysis | (PDF) | (PDF) |
In this experimental module you will study an unusual protein, one that allows yeast to bind a metal, gold. Over the next few weeks you will purify yeast based on this binding property, and then you'll vary some experimental condition to improve the yeast/gold interaction. Using your optimized conditions, you will screen a library of yeast to isolate a new gold-binding strain. The DNA encoding your new gold-binding protein will be sequenced and, with any luck, you'll elucidate some amino acid requirements for the yeast/metal interaction.
