Team:UCLA/Notebook/Recombinant Expression/21 May 2015

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• Starter culture creation
  • Looked at plates streaked from [tubes Tokyo gBlock 3/4/5]
  • Each plate had a good amount of isolated colonies, well streaked.
  • Observation of the streaked colonies under gel box UV yielded several colonies with high fluorescence, enough to verify proper insert presence.
  • Colony morphology was also identical, which indicates lack of satellite colonies.
    • Picked an individual colony and in separate tubes prepared starter and inoculated 10mL LB supplemented with 1x Amp. Placed in 37 deg shaking at 1600. Will check cell growth at 0900 tomorrow]

• Zymo Mix N' Go Competency tested
  • Yesterday's 50mL culture of BL21(DE3) streaked from Mark Arbing and starter cultures made were grown to OD600 of 1.2 -- way above the 0.4-0.6 range recommend by Zymogen.
  • Competency was conducted regardless, resuspending cell pellets in competency buffer wafter wash buffering the cells from each culture.
  • David aliquotted the resulting DH5alpha and BL21(DE3) cells into 50uL samples, and froze in -80.

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Replating of a glycerol stock of a Tokyo G-Block aliquot #5. Cells were scraped from the stock and streaked onto an LB agar/Amp plate, and incubated overnight at 37 degrees. Single isolated colonies are visible after the 4th streak; cells fluoresce under UV box illumination

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Replating of a glycerol stock of a Tokyo G-Block aliquot #4. Cells were scraped from the stock and streaked onto an LB agar/Amp plate, and incubated overnight at 37 degrees. Single isolated colonies are visible after the 4th streak; cells fluoresce under UV Box illumination

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Replating of a glycerol stock of a Tokyo G-Block aliquot #5. Cells were scraped from the stock and streaked onto an LB agar/Amp plate, and incubated overnight at 37 degrees. Single isolated colonies are visible after the 4th streak; cells fluoresce under UV Box illumination

Julian's Work

• Today I did Cell Lysis
• I thawed the 30C pellets from the freezer. I followed the nature protocol for all cell lysis with some differences as noted below. <a href="http://www.nature.com/nprot/journal/v4/n3/abs/nprot.2008.250.html"></a>
• Resuspended the pellet in 1X Lysis buffer (See protocol). I added 3mL of buffer/g of cells for a total of 8mL. Mixed to homogeniety and to break up pellet.
• I initially added a small amount of chicken egg lysozyme into the cells and incubated for 20 min. I then added 0.5mL Dnase and again incubated for 20 min. The mixture looked somewhat viscous and was completely homogenous. Cells appeared to have lysed. I also added PIC mixture found in the fridge near rocky's bench.
• I then centrifuged at 5000g for 25 min to pellet the insoluble stuff. This formed a hard pellet and the supernatant was no longer clear.
  • However, this pellet looked a lot like the starting cell pellet so I was not sure if lysis had taken place.
• I tried to lyse the cells further by adding another 1mL of 8X lysis buffer. I added more lysozyme as well and incubated for a further 30 min.I repeated the above steps and reserved this supernatant separately. Final pellet looked somewhat better and more lysed. This second supernatant was frozen.
• For the 1st supernatant, I heated at 80C for 10 min using the heat block. This caused a white protein precipitate to form. The protocol says this step will precipitate many endogenous proteins but leave the silk proteins in solution. I then centrifuged as directed in the protocol to pellet the precipitant.
  • the supernatant still had some precipitant floating in it after centrifugation
  • this was removed by syringe filtration
• final produce was clearer than before precipitation but not entirely clear indicating proteins were still present in the sample. This was also frozen at -80C

Next Steps

• Once the concentrators come in I will use them to concentrate the supernatant down to run over the column.
• I will purify the supernatant via IMAC
  • will try to use a vacuum to speed up the process
  • will run SDS page to verify the silk is there as expected and that purification was good

--Jlutze 18:33, 22 May 2015 (CDT)