Team:Wageningen UR/Notebook/Journal/VLP Generation
VLP Generation
By: Niek Savelkoul
Here you can find Niek's work on the production of Virus-Like Particles (VLPs) from various viruses. Curious about his results? Read more about it here.
May
Week 1 (1st of May - 7th of May)Got our own office. Started Literature work on experiment design.
Week 2 (8th of May - 14th of May)
Worked on Thesis proposal and overall in silico design of my experiment. Went to BCF career event in Utrecht.
Week 3 (15th of May – 21st of May)
Worked on Thesis proposal some more and overall in silico design of my experiment.
Week 4 (22nd of May – 28th of May)
Finished proposal, ordered primers and started cell-culture work. Maintained my SF-21 cell line.
June
Week 5 (29th of May – 4th of June)Started with labwork: Performed RNA extraction of MAYV from a Trizol sample.
Week 6 (5th of June – 11th of June)
One step reverse transcription PCR of MAYV structural cassette. First RT-PCR failed, unfortunately.
Week 7 (12th of June – 18th of June)
One step RT-PCR was tried again, but failed once more. Strategy was thought over and new RNA was isolated. Changed approach: I now plan to first synthesize cDNA from the MAYV-RNA.
Week 8 (18th of June – 24th of June)
cDNA generated, successful Phusion PCR with attB overhangs. Started Gateway cloning.
July
Week 9 (25th of June - 2nd of July)Gateway troubleshooting, tried different conditions but to no avail so far.
Week 10 (3rd of July - 9th of July)
Found a mistake in the BP gateway reaction, have to start over again.
Week 11 (10th of July - 16th of July)
Successful BP reaction, started different approach to directly ligate to pFastBac: I cut the vector and insert with the correct restriction enzymes, gel purified and ligated the fragment into the vector.
Week 12 (17th of July - 23rd of July)
BP correct, started with LR. Checked pFastBac ligation, correct. LR also correct. Sent insert for sequencing. Started with Bac-to-Bac.
Week 13 (24th of July - 30th of July)
Found a mutation due to wrong primer in the C-terminus. Decided to repeat experiments, but due to the small mutation continue with the Bac-to-Bac. Re-amplified insert with correct primers, ligated and checked with colony PCR. First transfection into SF-21 cells with MAYV-F and MAYV-D construct. Ordered primers for sequencing complete insert.
August
Week 14 (31st of July - 6th of August)Monitored transfection, sequenced complete insert. Infected Sf-9 shaker cultures with AcMNPV-ZIKV, AcMNPV-CHIKV. Samples prepared for ultracentrifugation.
Week 15 (7th of August - 13th of August)
Sample of CHIKV and MAYV VLP’s were ultracentrifuged in sucrose gradients. New infection for production of MAYV-VLP’s in Sf-9. The new pFastBac construct was checked using restriction analysis and sent for sequencing.
Week 16 (14th of August - 20th of August)
MAYV-VLP’s purified using sucrose gradient in ultracentrifuge. SDS/Western blot was run on purified samples of ZIKV, MAYV and CHIKV VLP’s.
Week 17 (21st of August - 27th of August)
Sequencing of new pFastBac correct, continued with Bac-to-bac.
September
Week 18 (28th of August - 3rd of September)New SDS/Western blot on MAYV samples.
Week 19 (4th of September - 10th of September)
New infection of MAYV in SF-21, followed by protein analysis on SDS/Western blot. Started production of CHIKV spike proteins in SF-21.
Week 20 (11th of September - 17th of September)
Analyzed CHIKV spikes using SDS/Western blot. Have to redo the samples.
Week 21 (18th of September - 24th of September)
New infection of SF-21 with CHIKV spikes. Analyzed MAYV for the last time, confirmed the presense of MAYV-VLP’s in the insect cells.
October
Week 22 (25th of September - 1st of October)Infected new SF-21 cells with CHIKV-spikes.
Week 23 (2nd of October - 8th of October)
Analyzed spikes after washing, according to phage display protocol. Spikes stick to sepharose beads. Started production of more CHIKV spikes in Sf-21.
Week 24 (9th of October - 15th of October)
Produced more CHIKV spikes. Helped to produce more helper phage.
Week 25 (16th of October - 22nd of October)
Helped with setup of Phage display.
Week 26 (23rd of October - 29th of October)
Started with phage display panning.
