Team:Wageningen UR/Notebook/Ben
Sensing Xylella fastidiosa
By: Ben Kuipers
In Ben his labjournal, he detailed his journey to built a sensing circuit that could recognize X. fastidiosa's quorum sensing molecule DSF. The results of his journey can be read on his results page.
May
Week 1 (13th of May – 19th of May)
Started the literature study.
Week 2 (20th of May – 26th of May)
Continued literature study. Visited the BCF career event.
Week 3 (27th of May – 2nd of June)
Continued literature study and started to design.
June
Week 4 (3rd of June – 9th of June)
More literature study!
Week 5 (10th of June – 16th of June)
The SnapGene journey started, this means that the lab journey is getting close.
Week 6 (17th of June – 23rd of June)
The lab journey started where I prepared media, antibiotic stocks and transformation buffers. E. coli DH5Alpha cells were made competent. The first parts from the iGEM repository were retrieved and E. coli cells were transformed with these.
Week 7 (24th of June – 30th of June)
Transformed colonies were picked, re-streaked, and liquid cultures were made for miniprepping and glycerol stocks. Continued with designing all the parts necessary for my project.
July
Week 8 (1st of July – 7th of July)
Looked into different types of protocols on how to treat certain bacteria.
Week 9 (8th of July – 14th of July)
Cell lysate from X. campestris was retrieved and DNA was isolated. The specific primers to amplify the C-terminal part of the histidine kinase gene rpfC were used to amplify this part. The ordered gBlock of the X. fastidiosa N-terminal rpfC was dissolved and used in PCR.
Week 10 (15th of July – 21st of July)
The N and C termini of the rpfC gene were cloned together in the pSB1C3 by GoldenGate assembly and transformed in DH5alpha. The initial attempt failed, second attempt after repeating GoldenGate worked. Plasmids containing the chimeric rpfC (rpfCch) and rpfG were minprepped and send for sequencing.
Week 11 (22nd of July – 28th of July)
Finalized the complete design of my project for the upcoming weeks.
Week 12 (29th of July – 4th of August)
The rpfCch and rpfG were amplified from the backbone. The riboswitch was ordered as a gBlock, dissolved and used in PCR. The Anderson promoter J23100 was ordered as two primers and annealed to get it as a part. A GFP construct was retrieved via the lab. The J23100 promoter, rpfCch and rpfG were ligated via GoldenGate and transformed. The riboswitch and the GFP were ligated and transformed. And again, 160 chemical E. coli competent cells were prepared.
August
Week 13 (5th of August – 11th of August)
The ligation of promotor+rpfCch+rpfG as one part (with and without backbone) failed time after time. The Vc2-riboswitch and the GFP were sent for sequencing. Also, a diguanylate cyclase, phosphodiesterase and ompR promoter were retrieved from the iGEM repository plates.
Week 14 (12th of August – 18th of August)
New strategies for getting the promoter+rpfCch+rpfG was conducted, still negative results. The Vc2-riboswitch was also ligated in a different backbone, successfully. Also, the design was started for a second strategy where the X. fastidiosa rpfC N-terminal part would be fused to the EnvZ C-terminal part of E. coli.
Week 15 (19th of August – 25th of August)
Gave myself an undeserved holiday.
Week 16 (26th of August – 1st of September)
After one week of soul searching, I decided to first PCR the rpfCch with the verification primers to get less probable aspecific binding of the primers. Now the RBS and promoter were added in two PCRs.
September
Week 17 (2nd of September – 8th of September)
The rpfC+rpfG and the rpfC/EnvZ were ligated and transformed and sent for sequencing. The OmpR promoter and GFP were ligated as one part. The EnvZ K.O. strain JW3367-3 was retrieved from the Keio Collection plates and the KanR gene was removed from the strain in one week.
Week 18 (9th of September – 15th of September)
Several constructs were amplified, ligated and transformed to make several combinations of systems. Of course, every transformed failed. The rpfCch+rpfG and Vc2+GFP plasmids (2 plasmid system) were treated with the clean and concentrator for a collaboration with Marijn to test this system in-vitro with TXTL, with and without DSF.
Week 19 (16th of September – 22nd of September)
Checked if the EnvZ K.O. lost the KanR gene, this was successful. A double transformation of the 2 plasmid system was executed, successful. Incubated with and without DSF, no visible fluorescence. The rpfC/EnvZ and OmpR promoter+GFP were ligated. The rpfCch + Vc2/GFP (1 plasmid system) was ligated and transformed in different backbones, successful. TherpfCch construction was repeated for different backbones.
Week 20 (23rd of September – 29th of September)
Went to the Safety workshop “SafeChassis”. rpfC/EnvZ+ompR promoter/GFP ligations were amplified and transformed. Also the phosphodiesterase (YhjH) was amplified with RBS and promoter and transformed. Also, the backbones were amplified for the incorporation of the Anti-CRISPR of Alex (replacing the GFP in my system). The GFPs and rpfC (TMD) were amplified and fused to see if the rpfC is getting transcribed and transported to the membrane, which is the case.
Started wiki writing.
Week 21 (30th of September – 6th of October)
PdeH was sent for sequencing, which had a very odd mutation, this added 20 bp extra between RBS and start codon. The rpfC/EnvZ+ompR promoter/GFP showed odd results in colony PCR. Sent for sequencing, rpfC/EnvZ gene was deleted from the construct. Dropped this part due to time constraints. Re-amplified the PdeH for transformation.
Wiki writing.
October
Week 22 (7th of October – 13th of October)
Guess what, PdeH had the same mutation as last week, also dropped this part due to time constraints. Colony PCR on the Anti CRISPR constructs was odd, sequenced it, no Acr present in the contructs. Also dropped this part. As a final experiment we decided that qPCR can be used as a fast tool to see if the riboswitch + GFP are getting transcribed, but not translated (as we are not able to turn it on yet). So RNA extraction was carried out twice, once with the Maxwell robot and once with the RNeasy kit, Qiagen. Both yielded DNA contamination, so not useful for cDNA preparation and therefore qPCR.
Even more wiki writing.
Week 23 (14th of October – 20th of October)
Treated the RNA sample again with DNase. This will be the last experiments in the lab, sad face. Almost only wiki writing.