UCLAiGEM:Notebook/Organization/2015/04/17

From OpenWetWare

Jump to: navigation, search
Project name Main project page
Previous entry      


Spring week 3 General Meeting

Administrative

  1. Committees
  1. Financial
  1. SPARK launch date finalized for July 13th
  2. We need to write up a detailed budget proposal for the $5000 we’re trying to raise.
  1. Using the budget for reagents and lab supplies.
  2. We’ll schedule a conference call with the Spark people afterwards
  1. SGI -- How much free synthesis, gibson reagents? Protein cage team have an idea of how much they’ll be using?
  1. Will offer some reagents, potentially some Gibson related materials.
  1. Any other groups still designing oligos/gBlocks?
  2. Extra fundraisers
  1. Donuts, etc.
  2. Fundrager, perhaps.
  1. Lab Management
  1. 7731 is ok for us to use now -- should we move our equipment over?
  1. Schedule a day for people to move our stuff over to 7731
  1. Next Monday, April 20th, at 1:00pm.
  1. Reminder: for now, no bacterial work allowed in 7731.
  2. I talked to a grad student from the Grundfest lab, he gave a tentative OK for us to use their bacteria shaker/incubator
  1. This grad student is also currently using 7731
  2. Email Jesse about this incubator.
  1. Info media
  1. Our social media has to improve in the next few months
  2. We’ll be giving everyone access to our Twitter account
  1. Still need a centralized person to oversee it.
  1. Megan will be adjuncting into our committee
  2. Will be ordering T-shirts on Monday!!!!!
  1. S - 4
  2. M - 8
  3.  L - 5
  4. Carter
  1. Need to update our igematucla.com website. Each project team create a page giving a synopsis of the field/context and project goals.
  1. Honeybee silk, functionalization, protein cages needs an update
  2. send a blurb to Anuved via email by Monday, 5 pm (20 April)
  1. Communications (Michael)
  2. Social Coordinator/Event Planner
  1. Slack
  2. Cricket, next Sunday, 11am.  (4/26)
  3. May 1st, Sri’s happy hour if you’re over 21 or especially have a fake (jk)
  1. Recruitment/Training
  1. Document training / recruiting from previous quarter
  2. Google Drive, program structure folder, summarize what we did Fall and Winter quarter.  Use a template for this information.  Feedback system.  Contact Nithin for Slack.  
  1. Outreach/Publicity
  1. Contact Aalhad for a Daily Bruin article right before our Spark campaign.  
  2. Reach out to UCLA Newsroom (Sri will contact Stuart Wolpert).
  3. Collaborating with Thunderclap
  1. send an email to Penny
  1. Time/Scheduling
  1. Create some sort of spreadsheet/ calendar to show who is in lab and when.  (for Jessica)
  1. Any other administrative announcements
  1. Idk how you guys have been documenting all your project stuff so far, but until we figure out a better system, please put everything into the “project team” folder in the Google drive.  
  2. I typed out a tutorial for exporting Google Docs to OpenWetWare.
  1. Please note that images will not be properly exported using this method.
  2. Also, in these meeting notes, please use Google Doc’s native outline formatting tool! It looks like this:

Project updates

  1. Cages
  1. Meeting with Yeates on Wednesday 11AM. Had discussions on preliminary papers and are setting up an agenda for the meeting.
  2. Talking Points: https://drive.google.com/open?id=1OUFKKXoWr0sEX0IDTZffHLOxJ_Nyml6NMAHXFkwoL78&authuser=0
  3. Introduce ourselves, how are program is structured, what our program timeline and project is like.  Ask what is feasibly possible, potential ideas, protease induced release, drug targeting, novel cage designs for biobricks, etc.
  4. Cage applications at the end would be very good for us to talk about today.  
  5. Andreas Martin
  1. Functionalization
  1. This week we isolated cells from the cultures grown last week and performed a standard alkaline lysis to collect the lysate extract (4x 10mL samples).  Extracts were frozen in -20 and the Affinity Column was prepared.  
  2. The first round of purifications were conducted today.  One issue is that we have a LOT of proteins, which would require doubling of the standard protocol and would use more buffer.  Also would run into the issue of diluting the sample far below the conditions needed for a good Western Blot signal or coomassie stain.  
  1. Could try using less elution buffer.
  2. Could try concentrating the sample down
  3. Could try packing with less resin BV.
  1. Need to purchase a ring stand, tubing for the affinity column, get Bradford assay kit, come up with a source for standardization of the reaction (BSA).
  2. Protein yields quantification.  Coomassie staining and using ladder, loading control, cell lysate, and purified proteins at different elution concentrations.  
  3. Microplate analysis of cell lysates would be a great starting point for a first experiment.  What we will first do is verify Tamura purity using SDS/Westerns, then do a fluorescence microplate analysis to verify.  
  4. Julian created additional constructs of Tamura in pSB1C3, and amplified the ABD gBlock.  Once we get enough yield from the PCR, we will do the Silver fusion to ligate the ABD and displace the GFP in the pSB1C3 Tamura construct.  After sequence verification and colony PCR, we will re-transform and express the ABD, and perform downstream experiments from there.  

  1. Materials
  1. Completed dialysis
  2. Will purchase dialysis tubing and PEG 10k for concentrating
  3. Will contact Seidlits or Wu regarding use of their lyophilizer
  1. Density, concentration of silk in gel, porosity, diffusion constant, stiffness, fluorescence assays for Tamura, thermostability (degradation rates)
  1. read into Wu’s papers
  1. Our group is thinking about possibly designing constructs for functionalizing other materials aside from threads
  1. Do we have the budget/manpower for this, or should the materials group just split up and join the other groups after we’re familiar with the protocols?
  2. read into Silk Elastin-like proteins
  1. Honeybee silk
  1. https://benchling.com/s/p7bClpzQ/edit
  1. G block just ordered.
  2. Primers designed to amplify out various things as well put it into (the primers have been designed, but we’ll order them as we need them)
  3. If time, go over the conflict with pET14b found in the paper https://www.embl.de/pepcore/pepcore_services/strains_vectors/vectors/pdf/pET-14b_map.pdf
  1. Next on the docket (While waiting for Gblock)
  1. Plan out purification scheme with just silk protein in order to hammer out the protocol. (Does not use tag purification, so will be a modifed protocol from the one the other functionalization groups are doing)
  2. Figure out all materials necessary and make a detailed wishlist as well as work flow (The paper uses BugBuster lysis kit for ex.)
  3. Here is the paper that details the protocol:
  4. Think about SpyTag, and making a construct asso. with SpyTag (SpyTag-GFP/Albumin)
  1. Sometime late next week, amplify out silk, and clone the construct into the biobrick backbone, as well as hopefully an expression vector.  
  1. Contact Dr. Arbing to request vector. (pET 14b hopefully)
  2. Think about adapting Tamura experimental designs to do the work much easier and with good quality data.  

  1. ICA/Ishmael
  1. Made all MaSp 2 constructs
  2. Still making more due to low yield after gel purification; will use a new kit
  3. Plan for next week
  1. Clone all MaSp2’s into psb1c3 and do transformation
  1. T4 ligase, T7 ligase, Taq DNA ligase, test different buffer combinations, different temperatures, different temperature “ramp-down” protocols.

Personal tools