Team:Wageningen UR/Notebook/Cleo
Triggering the Plant Immune Response
By: Cleo Bagchus
This is the notebook of Cleo. Here you can find more information on her journey from failed protein purification experiments to plant experiments. She worked on activating a plant immune response. This link will bring you to her results page.
May
Week 1 (13th of May – 19th of May)
We got our own office and started working full time. Divided all the subprojects among the team members.
Week 2 (20th of May – 26th of May)
Worked on proposal.
Week 3 (27th of May – 2nd of June)
Worked on proposal, had a meeting with Klaas Bouwmeester to discuss feasibility project and a collaboration. Joined the Biology Olympiad.
June
Week 4 (3rd of June – 9th of June)
Finished proposal.
Week 5 (10th of June – 16th of June)
Made experimental design, ordered primers and purified plasmid backbone. Joined the European meetup organized by the Rathenau institute.
Week 6 (17th of June – 23rd of June)
Gblock arrived containing three possible MAMP candidates, extracted the fragments.
Week 7 (24th of June – 30th of June)
DNA constructs for the protein purification of flg and NLP successful.
July
Week 8 (1st of July – 7th of July)
Successful formation of Hax construct and made electrocompetent cells.
Week 9 (8th of July – 14th of July)
Ligation and transformation of cells of three MAMPs. Sequencing results for NLP, flg and Hax successful.
Week 10 (15th of July – 21st of July)
Performed small scale purification of TEV protease and flg. No flg was visible on a 16% tricine SDS page gel.
Week 11 (22nd of July – 28th of July)
FPLC purification TEV protease.
Week 12 (29th of July – 4th of August)
Ran OD-corrected pre and post induction samples of cultures with flg on a plasmid, induced with different levels of IPTG on a 16% tricine SDS page gel. No difference in expression levels.
August
Week 13 (5th of August – 11th of August)
Ran FPLC purification flg. This was not successful.
Ordered primers for an MBP-MAMP fusion protein construct. Started cloning the different constructs.
Week 14 (12th of August – 18th of August)
Transformation of constructs failed (no colonies present). Increased the concentration of DNA for the second transformation.
Week 15 (19th of August – 25th of August)
General troubleshooting cloning. Finally performed extra cleanup steps to decrease salt concentration.
Week 16 (26th of August – 1st of September)
Sequencing of MBP-NLP and MBP-flg constructs successful. Performed TXTL of MAMP. This was not successful. Started growing plants. Ran pre/post induction samples of MBP-flg and MBP-NLP on 16% tricine SDS page gel. Indication of expression.
September
Week 17 (2nd of September – 8th of September)
Sequencing of MBP-hax construct successful. Tabletop purification of MBP-NLP construct.
Week 18 (9th of September – 15th of September)
Performed purification of an MBP-NLP protein construct using FPLC. This was successful.
Week 19 (16th of September – 22nd of September)
Attempted to purify cleaved NLP from an MBP-NLP construct treated with TEV protease using FPLC. No protein was detected on a 16% tricine SDS page gel.
Week 20 (23rd of September – 29th of September)
Stopped with protein purification work. Inoculated first batch of plants with Xanthomonas campestris campestris.
Week 21 (30th of September – 6th of October)
Waited for the second batch of plants to show symptoms. After not showing symptoms for a week, removed these plants. Introduced X. campestris in the second batch of plants.
October
Week 22 (7th of October – 13th of October)
Measured the lesion size on the second batch of plants. Inoculated the second batch of plants. Harvested plant material.
Week 23 (14th of October – 20th of October)
Performed analyses on the harvested plant materials.