iGEM Yale

Week 4: June 5-12

  • Monday:
    • Made glycerol stock of Canada cells.
    • Made plates 1mM IPTG + Amp
    • Made 1 L LB, completed 1/40 dilution of ZeAFP (added Amp), shook for 3 hrs, checked OD until .78 OD reached
    • Divided 1 L into 6 separate 100 mL solutions, half with IPTG, induced for 5 hours
    • Centrifuged 4 100 mL solutions at 4700 rpm for 15 minutes at 4 degrees celsius, saved supernatant/pellet for 2 (frozen at -20); for the other 2, we completed the following protocol:
      1. Washed with 50 mL water
      2. Resuspend in 100 mL ice-cold water
      3. Divided 1ml aliquots into 8 eppendorf tubes (half induced half uninduced)
      4. Added 100 and 10ul to IPTG and non IPTG plates (4 total) and grew at room temperature. Froze eppendorft tubes. Tomorrow we will unfreeze them for 6 hours on ice, then 1 hour at 4C, then replate to compare colony growth - essentially a survival assay after frozen : before frozen number of viable cells (this is obviously just a test assay)
    • Plated 6 samples of the remaining 2 100 mL solutions, using 10 and 100 microliters on IPTG, LB, Amp plates and non-IPTG, LB, Amp plates and stored 1 of each concentration in 0 degrees, 4 degrees, and room temperature
  • Tuesday:
    • Finished and presented project defense presentation to Sackler Institute and our advisers.
    • Made 3L worth of amp+iptg plates for large assay
    • Obtained TCA protein precipitation protocol: Stock solution 100% w/v Trichloroacetic acid (TCA). Recipie: dissolve 500g TCA (as shipped) into 350mL dH2O, store at RT. Protocol: add 1 volume of TCA stock to 4 volumes of protein sample. I.e. in a 1.5ml tube with maximum vol, add 250ul TCA to 1.0mL sample. Incubate 10min at 4C. Spin tube in microcentrifuge at 14K rpm, 5 min. Remove supernatant, leaving protein pellet intact. Pellet should be formed from whitish, fluffy ppt. Wash pellet with 200ul cold acetone. Spin tube in microcentrifuge at 14K rpm, 5 min. Repeat steps 4-6 for a total of 2 acetone washes. Dry pellet by placing tube in 95C heat block for 5-10min to drive off acetone. For SDS/PAGE, add 2X or 4X sample buffer (with or without BME) and boil sample for 10 min at 95C heat block before loading sample into Polyacrylamide gel.
    • Bacterial colonies for test of freezing assay are not growing yet - we will give more time b/c they were at room temperature previously.
    • Put induced and not-induced at 10 and 100ul on plates (the ones after frozen).
    • Prepared 50 mL SDS sample buffer: 50 mM Tris-Cl (pH 6.8), 2.5 mL; 100 mM DTT (made one 1 M stock solution); 2% (w/v) SDS, 1 g; 0.1% bromophenol blue, .05 g; 10% (v/v) glycerol (5 mL)
  • Wednesday:
    • Lawn of bacteria growing on RT plates.
    • OD measurements of frozen tubes to compare. Induced tubes have lower OD on average (statistically significant), likely due to protein expression. Need to compare % survival instead.
    • Interesting phenomenon whereby cells with anti-freeze protein in it forms elongated ice chunk while melting, but non-induced solution is fatter chunk. This is observed in eppendorf tubes containing the cells, but also in unfreezing supernatant.
    • Learned how to set a gel: do 2 at a time. Kit is in drawer. Add grey strips. Scrub glass holder and surface glass with wire/warm water. Dry spacers, ethanol, dry again. Put clean face of surface glass on glass. Have biorad facing you at top. Make sure that there is a tight seal, do on hard surface. Close the press while the two are sealed together. Then make separating buffer from stock solutions. Use 1/2 listed volumes to make 2 gels! After 10mls added add 40 microlitres of APS, then add 20ul TEMED (starts to polymerize right away). Add with pasteur pipet until it is right above the line. Stop below green. Then add water saturated butanol to the top, and let sit 50 mins.
    • Prepared three samples for SDS:
      1. supernatant with protein concentrated via 3000 m.w. filter conc. columns (Millipore MWCO Conc. Columns) - Pipetted 15 mL of supernatant into column, spun at 4000 g for 15 min at 4 deg C; then poured out flow-through and repeated with another 15 mL of supernatant until 50 mL of supernatant was used; then centrifuged one last time - With 500 remaining microliters, then added 200 microliters sample buffer and mixed thoroughly by hand
      2. supernatant with protein ppt'ed via TCA precipitation protocol
      3. pellet lysed/diluted in loading buffer, all from Monday night's centrifugation, and with and without IPTG)
  • Thursday:
    • Prepared Gels. Did TCA precipitation on supernatant of ZeAFP samples. Also concentrated supernatant using 3000 m.w. filters. Spun for 15 minutes at 4oC, 4000g. Repeated until concentration went down to 500uls. Added 200ul of sample buffer to this.
    • Insert pic of gel
  • Friday:
    • Prepared competent BL21/origami cells
    • Purchased primers for PCR amplifcation of ZeAFP, RiAFP and TmAFP
    • Innoculated 100 mL culture of ZeAFP-transformed cells
    • Resuspended cell culture pellets in lysis buffer (1.25mL EDTA/Tris buffer, pH 8.0, 50mM Tris. Add 0.128mL of PMSF from 1mM stock) and sonicated
  • Saturday:
    • Freeze tolerance assay prep: Harvest cells, washed once in 50mL H2O, resuspend in 100mL ice cold H2O, 1mL of suspension into microtubes, plate cells from tube, then freeze -20 overnight, thaw on ice for 6 hrs, incubate 4oC for 1 hour. Compare survival before and after.
  • Sunday:
    • 1 in 40 dilution of Tokyo Tech Cells, induction for 2 hrs with IPTG
    • Transformed competent cells with other TmAFP Plasmids
    • Counted cells from survival assay and OD assay (insert graph here)