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Week 2: May 22-29

• Monday:
  • Meeting scheduled in the Med School TAC building with Martin and Girish tomorrow at 2:30pm about using their plasmids with the anti-freeze glycoprotein that they isolated.
  • Meeting scheduled for next Wednesday with Farren at 2:30pm in West Campus, building B-31, Room 209.
  • Made TB buffer (DH5-alpha competent cell buffer). 5mLs of 1M HEPES buffer (final concentration 10mM), 75mL of 0.1M CaCL2*2H2O (final concentration 15mM), 9.32g KCl (final concentration 250mM) was added to a beaker. Volume was brought to 300mL. pH was brought to 6.7 with 6.25M KOH. 5.44g of MnCL2*4H2O (final concentration 55mM) was added, and the volume was brought to 500mL. The contents of the beaker were sterile filtered into a sterile bottle purchased from the basement store.
  • Made 500mL LB buffer (no agar), which was distributed in 2x250mL Erlenmeyer flasks and 2x500mL Erlenmeyer flasks. No antibiotic was added.
  • Added 250ul of frozen DH5-alpha cells to 50ml culture flask. Incubated on shaker at 37 overnight.
• Tuesday:
  • Transferred 4mL of overnight culture to 200mL tube. Waited 5 hours before checking the OD. The OD was >2 but it should be 0.55. Will innoculate another 50mL culture overnight but wait shorter amount of time.
  • Results from meeting with Fikrig lab: They have not yet purified their iAFGP. They tried expressing it in E. coli and it did not work for them (likely because of the glycosylation sites). They have now expressed it in Drosophila cells and have been working on purifying the protein for about half a year. They cautioned against using anti-freeze glycoproteins for our project. They said that they would be willing to help us with assays for our project. They have experience with ice recrystallization assays, ice morphology assays, and a 4C cold tolerance assay in E.coli. They recommended contacting Peter Davies from Queen's University in Kingston, ON - he is willing to share plasmids from type III anti-freeze proteins. They asked if they could use which ever AFPs we end up expressing and purifying as positive controls for their assays once they purify iAFGP glycoprotein.
• Wednesday:
  • Possibility of using a new AFP sequence in our project instead of one that has already been expressed in E. coli was investigated. Obviously we need to be able to defend a decision to do this, and not just express it because it is new. One possibility is using a hyperactive thermolabile AFP that was recently cloned from fish but not yet expressed in E. coli. This anti-freeze protein has exceptionally high TH activity but it irreversibly denatures at room temperature. This is a useful property, as denatured proteins degrade much faster (public more amenable to this if we are using it in food / on sidewalks).
  • Discussed with one of the grad advisors whether using MAGE to optimize the general secretory pathway in the EcNR2 strain (inactivating relevant genes like proteases, optimizing RBS sequeneces of relevant genes, etc) would be a good direction to take the project. First, we would need to find a readout. This might be GFP (especially if the Tokyo Tech part works), or we might use the cold tolerance assay (see if more grow at lower temperatures). Might be better to buy the GFP+AFP, then take GFP off with primers that are complementary to AFP plus a new restriction enzyme site. It seems like for our purposes we would be using MAGE to optimize the general secretory pathway in the EcNR2 strain (inactivating relevant genes, optimising RBS sequences of relevant geness, etc), and not tinkering with the AFP protein itself. We wondered how the optimized secretory pathway would be submitted to iGEM as biobricks. Would probably have to PCR all of the locuses that we target with MAGE, put them in plasmids, and submit them. Also discussed possiblity (as an addition to the experiment) of expressing AFP in fungi (tricoderma/neurospora).
  • In the morning 4mL of the overnight 50mL culture was transferred to the 200mL flask. OD was checked ever 30 minutes. After 1 hour and 45 minutes the OD was at 0.588 (ideally should be 0.55). Cells were transferred to 6 sterile falcon 50mL polypropylene tubes (35mL put in each tube). Tubes were stored on ice for 10 minutes. Cells were centrifuged in Strobel lab upstairs at 2500xG for 10 minutes at 4C. Medium was poured off and tubes were stood upside down on paper towel + kip wipes for 1 minute to remove remaining medium. Pellets were resuspended in ice cold TB buffer. 10mL buffer was added to all 6 of the tubes to resuspend the cells. Then the tubes were combined to make 2x 30mL tubes. The two tubes were centrifuged at 2500xg for 10 minutes at 4C to recover the cells. Medium was poured out, and tubes were stood upside down on paper towel for 1 minute to remove remaining medium. Pellets were resuspended by gently pipetting in a total of 20mL of ice coldTB buffer (2x10 mL). Cells were combined in single tube. 1.5mL DMSO was added and tube was mixed by swirling. Tube was stored on ice for 10 minute. 100 microlitre aliquots were put into chilled eppendorf tubes. Tubes were closed tightly and flash frozen in liquid nitrogen and stored in the -80 freezer to the bottom left.
  • Two tubes were set aside after being flash frozen. Contamination was tested by plating untransformed cells on an Amp plate. Competence of cells was tested by transforming a "control" plasmid containing Amp. 1ul of plasmid was added to 50microlitres of cells in a chilled 14mL bacterial culture tube to look at competence, and 1ul of ddH2O was added to 50 microlitres of cells in a chilled 14mL bacterial culture tube to look at contamination. Tubes were incubated on ice for 30 minutes. Cells were heat pulsed in a 37 bath for 45 seconds, then put on ice for 2 minutes. 450microliters of LB buffer was added to each tube, and tubes were incubated at 37C for 1 hour while shaking. 100uL of each culture was spread on an Amp-LB/agar plate and was put in the 37C incubator.