Team:Wageningen UR/Notebook/Alex
Constructing the dCas9-Anti CRISPR gene circuit
By: Alexander Niederau
This subproject underwent quite some changes during the time. At the beginning, the idea was to work with Xylella fastidiosa and its phages themselves! Unfortunately, we could not receive the phages... Around the same time, we started developing the idea of a Phage Delivery Bacteria. So it came to pass that I switched to constructing the dCas9-Anti CRISPR gene circuit. If you want to know the outcome of this subproject, read the results page .
May
Week 1 (13th of May – 19th of May)
Getting started, diving into literature about X. fastidiosa and its phages, requesting the phages from the US.
Week 2 (20th of May – 26th of May)
Even more literature, planning of phage engineering, setting up collaboration with a lab having experience in X. fastidiosa work, waiting for reply regarding the phages.
Week 3 (27th of May – 2nd of June)
Proposal writing, experimental setup planning, still waiting for reply regarding the phages...
June
Week 4 (3rd of June – 9th of June)
Waiting... and supporting my team members on plant related experiments.
Week 5 (10th of June – 16th of June)
Giving up hope to ever receive the X. fastidiosa phages... thinking about a chassis to deliver our Xylencer phage.
Week 6 (17th of June – 23rd of June)
Diving into literature about gene circuits, CRISPR interference and Anti-CRISPRs.
Week 7 (24th of June – 30th of June)
In silico planning: choice of Anti CRISPR, design of gBLocks and primers, experimental set up.
July
Week 8 (1st of July – 7th of July)
Proposal writing and first cloning steps in the lab to obtain an inducible dCas9-Anti CRISPR gene circuit targeting GFP.
Week 9 (8th of July – 14th of July)
Primer reparation, PCRs, gels, extractions, Gibson reactions and transformations...
Week 10 (15th of July – 21st of July)
First sequencing results showed mutations in the single guide RNA and the terminator of dCas9. Coincidence or caused by toxicity?
Week 11 (22nd of July – 28th of July)
Repetition of PCRs, gels, extractions, Gibson reactions, transformations and sequencing. Reoccurrence of mutations at the same positions hinted towards toxicity being the cause of the problem.
Week 12 (29th of July – 4th of August)
New approach: Usage of Restriction-Ligation to assemble gene circuit plasmids.
August
Week 13 (5th of August – 11th of August)
Sequencing results confirmed the integrity of the plasmids -> first plate reader assays could be performed!
Week 14 (12th of August – 18th of August)
Anti CRISPR shows repression of dCas9 and restoration of GFP signal -> the gene circuit works! However, the expression of the Anti CRISPR is quite leaky when not induced...
Week 15 (19th of August – 25th of August)
Randomization of the Anti CRISPR’s RBS site to lower basal expression and leakiness.
Week 16 (26th of August – 1st of September)
RBS mutant screening seemed promising! Sequencing of randomized RBS sites.
September
Week 17 (2nd of September – 8th of September)
Transfer of mutated RBS site into my plasmids to repeat my initial assay.
Week 18 (9th of September – 15th of September)
Parallel, construction of dCas9-Acr gene circuit via multiplex targeting of the bacteriophage lambda promoter PL and PR.
Week 19 (16th of September – 22nd of September)
Repetition of the assay of week 14 -> randomized RBS shows desired effect!
Week 20 (23rd of September – 29th of September)
Multiplexing plasmids were ready to use!
Week 21 (30th of September – 6th of October)
Plate reader assay with multiplexing dCas9-Acr gene circuit showed successful repression phage lambda promoter PL and PR controlling GFP and RFP expression.
October
Week 22 (7th of October – 13th of October)
Repetition of last experiments. Starting to summarize results and write pieces for the wiki.
Week 23 (14th of October – 20th of October)
Wiki!