Difference between revisions of "20.109(S24):M2D5"

Introduction

Protocols

Part 1: Induce expression of YSD peptide

For timing reasons, the induction steps were completed prior to class. So you understand how the cell suspensions you will use for the metal uptake assay were created, please review the steps below.

1. Inoculated 5 mL of SD-R media with a colony of M17 cells transformed with the pYAGA containing your peptide of interest.
2. Incubated the culture overnight at 30 °C with shaking at 220 rpm.
3. Dilute the overnight culture 1:10 in 10 mL of fresh SD-R media.
4. Incubate at 30 °C for 4 hours with shaking at 220 rpm.
5. To induce cell surface peptide expression, pellet cells and resuspend in 10ml of SD-G media.
6. Incubate overnight at at 30 °C with shaking at 220 rpm.

Part 2: Perform metal uptake experiment

1. Obtain M17 transformed with pYAGA_peptide culture from the front bench
2. While you prepare the experiment with your mutant, the instructors will prepare controls using the following cultures:
   • Untransformed M17
   • M17 transformed with control peptide
3. Gently tritruate with a 1ml pipette to create a homogeneous suspension for each culture
4. Obtain cuvettes from the front bench to measure the OD₆₀₀ of each culture prior to beginning the experiment
5. Add 1ml of each cell suspension to individual cuvettes and read on the spectrophotometer
   • Use 1ml SD-G for a blank.
6. Record these numbers in your notebook
7. Using SD-G media as a diluent, dilute each of your cultures to OD₆₀₀ ~ 1.0 and a final volume of 8ml. This does not have to be exact, but the cultures should have a similar OD₆₀₀ before you begin the experiment.
8. Add CdCl₂ and FeCl₂ for final concentration of 100μM in each culture and add to a new glass tube.
9. Incubate your labeled glass tubes shaking at 30°C for 2.5 hours to allow uptake.
   • During this incubation time, complete Parts 3 of the wiki.
10. Following incubation, take an additional OD₆₀₀ reading to account for any changes in culture density, and allow normalization of data across groups.
    • Record this in your notebook.
11. Transfer your cultures to 15ml conical tubes.
12. Centrifuge cultures at 1000 xg for 5 minutes to pellet cells.
13. During centrifugation, label 2 metal-free 15ml conical tubes for your mutant culture, and prepare a 10ml syringe filter for each culture
14. Using serological pipette, remove 6ml media supernatent without disrupting the pellet and add it to a metal-free conical tube.
15. Bring your samples to the front bench and add 175ul ultra-pure nitric acid to each sample. Return to your bench with your samples.
16. Using a serological pipette, carefully triturate each sample to fully mix the acid.
17. Open your 10ml filter syringe and place it over the top of a fresh metal-free conical tube. Add the mixed sample to the open syringe and use the plunger to push the sample through the 0.22 μM filter.
18. Close each tube with sample and place them at the front bench.
19. Discard materials used to filter the samples in the black bin at the front bench.

In your laboratory notebook, complete the following:

• What volume of CdCl₂ will you add to obtain a final concentration of 100 μM?
• What volume of FeCl₂ will you add to obtain a final concentration of 100 μM?
• Why is it important to use metal-free tubes to store your samples for analysis?
• Why is it important to filter your samples before submitting them for analysis?

Reagents list

• Synthetic dropout - uracil (SD-U) media: 0.17% yeast nitrogen base without amino acid and ammonium sulfate (BD Bacto), 0.5% ammonium sulfate (Sigma), 0.192 % amino acid mix lacking uracil (Sigma), 2% glucose (BD Bacto), 0.1% adenine hemisulfate (Sigma)
• Synthetic dropout - raffinose (SD-R) media: 0.17% yeast nitrogen base without amino acid and ammonium sulfate (BD Bacto), 0.5% ammonium sulfate (Sigma), 0.192 % amino acid mix lacking uracil (Sigma), 2% raffinose (Sigma), 0.1% adenine hemisulfate (Sigma)
• Synthetic dropout - galactose (SD-G) media: 0.17% yeast nitrogen base without amino acid and ammonium sulfate (BD Bacto), 0.5% ammonium sulfate (Sigma), 0.192 % amino acid mix lacking uracil (Sigma), 2% raffinose (Sigma), 2% galactose (Sigma), 0.1% adenine hemisulfate (Sigma)
• Cadmium chloride (Sigma), stock concentration= 100mM
• Iron chloride (Sigma), stock concentration= 100mM
• ULTREX II, Ultrapure Nitric acid (J.T. Baker)
• Hydrophilic PTFE 0.22μm filters (J.T. Baker)
• Metal-Free sterile polypropylene tubes (VWR)

Navigation links

Next day: Quantify cadmium removal from media