Presentation on theme: "Yeast gene expression lab using -galactosidase vectors that can be completed in one 2 hour laboratory session. Stephanie C Schroeder, PhD Assistant Professor,"— Presentation transcript:
Yeast gene expression lab using -galactosidase vectors that can be completed in one 2 hour laboratory session. Stephanie C Schroeder, PhD Assistant Professor, Dept. of Biological Sciences Webster University, St. Louis, MO
Background BIOL 3050/3051 is a 16-week sophomore level Genetics Lecture and Laboratory course. Both lecture and lab are taught by same instructor (me) The lab meets 3 hours, once per week. The lab classroom is shared between other classes, so is not accessible to students outside of the lab period.
Lab Schedule Title/TopicModel Organism Meiotic RecombinationSordaria Monohybrid/DihybridCorn/Drosophila/Yeast Sex Linked TraitsDrosophila Genetic LinkageDrosophila TransformationYeast (importance of CEN/ARS) Population GeneticsDrosophila DNA repair (UV mutagenesis)Yeast Mitochondrial DNAHuman Gene expressionBacteria and Yeast
Background This lab was designed to demonstrate two principles of gene expression in eukaryotes 1.Differences between positive and negative acting elements 2.Synergy with multiple promoter elements.
Title: Eukaryotic Gene Expression Purpose: To perform b Galactosidase assays on yeast strains containing plasmids with different promoters to determine the activity of these promoter fragments.
Strains were generated in lab of Tony Weil, at Vanderbilt University.
Constructs for Part One lacZ ABC T Wild Type lacZ ABC T ABC T x Deletion ElementA x Deletion Element B
Results from Original Paper Identifying Three Cis-Acting Elements A A B C
Part Two: Synergy Element C was subcloned into the XhoI site at copy 2 copies 3 copies 4 copies
Pre Lab Prep Week before : (I have students make the Z buffer and ONPG) 1. Streak out strains from glycerol stock onto Sc-ura plates 2. Make Z buffer, ONPG (wrap in foil), Na 2 CO 3 Day before lab: (I do) Inoculate 5 ml cultures of Sc-ura with strains Morning of lab: 1.Read Absorbances at 600 nm for overnight cultures 2.Aliquot yeast into 1.5 ml tubes for students 3.Aliquot SDS, Chloroform, Na 2 CO 3
Protocol For Students (They work in pairs) You have 8 tubes (labeled 1-8) which contain 0.2 mL each of the yeast strains listed in Table I. At the beginning of your lab session, I will tell you the A600 for each strain, to enter into your Table I. 1. Vortex the cultures to mix the cells.. 2. Add 0.8 ml Z buffer to each tube. (P-1000 set to 080) 3. Add 50 ul chloroform to each tube (P-200 set to 050) 4. Add 20 ul 0.1% SDS to each tube (P-20 set to 200) 5. Vortex each tube for 10" 6. Add 200 ul ONPG (1mg/ml in Z buffer +BME). Vortex the tubes to mix. 7. Time reaction at Room temperature (30 minutes from time added to tube 1) 8. Stop with 0.5ml 1M Na 2 CO Pellet 5 minutes in eppendorf centrifuge 10. Carefully transfer upper layer to a cuvette (chloroform will “ etch ” or dissolve the cuvettes) 11.Read the Absorbance at 420 nm (blank with tube that says Blank)) Taken from Cold Spring Harbor Yeast Genetics Lab Handbook (1989)
Cell cultureAbsorbance at 600 Volume (mls)TimeAbsorbance at 420Activity Units 1. No Promoter Wild Type Promoter Deletion DNA Element A Deletion DNA Element B CYC1 vector alone CYC1 + 2 copies element C CYC1 + 3 copies element C CYC1 + 4 copies element C Table I:
Calculate activity using the following formula: Activity Units= Absorbance at 420 nm x 1000 = Velocity/mL A600 x 30 minutes x 0.2 mL (200uL) Write-up/ Conclusions Construct ONE graph (bar graph) with the Activity Units for each construct on the Y axis and the names of the promoter constructs on the X axis. Use this graph to answer the following questions.
Activity Typical results from student experiments
Questions: 1.In the equation to determine the -galactosidase activity for our samples, why do we divide by the Absorbance at 600 nm? 2.What fold difference is there in -galactosidase activity between sample 1 (empty vector) and sample 2 (full length promoter)? 3.Do you think the DNA sequence Element A is a positive acting element or a negative acting element based on the difference between sample 2 (full length) and sample 3 (deletion of Element A)? Explain your rationale. 4.Do you think the DNA sequence Element B is a positive acting element or a negative acting element based on the difference between sample 2 (full length) and sample 4 (deletion of Element B)? Explain your rationale. 5.Samples 5-8 are constructs in which I inserted sequences from the TBP promoter into a DIFFERENT promoter (CYC1) to see their effect in a heterologous system. Based on the differences between samples 6,7, and 8 (2, 3 and 4 copies of Element C) compared to sample 5 (CYC1 alone), is the sequence Element C a positive or negative acting element? Explain your rationale 6.Based on your answers above, what do you think would happen if we put the sequences Element A from the TBP promoter into the CYC1 promoter? Based on the activity of the CYC1 promoter alone, do you think we could really measure a difference?
Assessment Classroom discussion (lecture and lab) Students answers to questions Ability to apply to lecture exam/cumulative final