Team:Wageningen UR/Notebook/Sebastiaan
Phage repression
By: Sebastiaan Kuiper
This is Sebastiaans notebook, where you find information on the repression of phage genomes using both dead and active Cpf1. His results can be found here.
May
Week 1 (13th of May – 19th of May)
Full-time iGEM has started. We took place in our office and started to dive deep into literature.
Week 2 (20th of May – 26th of May)
Literature research and the acquisition of potential sponsors for our project.
Week 3 (27th of May – 2nd of June)
Literature research and proposal writing.
June
Week 4 (3rd of June – 9th of June)
Proposal writing, literature research and acquisition of companies for sponsoring.
Week 5 (10th of June – 16th of June)
Designing experimental setup.
Week 6 (17th of June – 23rd of June)
Finalizing the experimental research setup and preforming in silico validation with SnapGene.
Week 7 (24th of June – 30th of June)
Ordering all the required plasmids, g-Blocks and oligo’s. Prepare for lab work by gathering and producing all required materials, media solutions and antibiotic stocks.
July
Week 8 (1st of July – 7th of July)
Started with labwork; growing bacteria, miniprepping, obtaining required plasmids and sending them for sequencing.
Week 9 (8th of July – 14th of July)
Co-transforming dCpf1 and the RFP-GRP target plasmids into E. coli. Making the co-transformed bacteria competent again and transform them with the targeting and non-targeting spacer plasmids.
Week 10 (15th of July – 21st of July)
Establishing a standard for the degree in inhibition of GFP by targeted and non-targeted dCPF1.
Week 11 (22nd of July – 28th of July)
Synthesizing new GFP-RFP plasmids with bacteriophage Lambda promoters (Lambda-GFP-RPF) and synthesizing an interchangeable, modular guide plasmid system for (d)Cpf1.
Week 12 (29th of July – 4th of August)
Screening for correctly synthesized plasmids by colony PCR and sequencing.
August
Week 13 (5th of August – 11th of August)
Production of specific spacer constructs for targeting bacteriophage lambda and therefore also our Lambda-GFP-RFP plasmid.
Week 14 (12th of August – 18th of August)
Screening for correctly synthesized spacers by colony PCR and sequencing.
Week 15 (19th of August – 25th of August)
Unfortunately, the spacers were not properly produced and therefore we had to redo the cloning of the spacers for this week.
Week 16 (26th of August – 1st of September)
Analyses of the spacers and the co-transformation of dCpf1 and Lambda-GFP-RFP plasmid constructs.
September
Week 17 (2nd of September – 8th of September)
We finally have all of our required plasmids! The dCpf1, Lambda-GRP-RFP and all of the (non-)targeting spacers are in a proper order.
The production of competent cells, one of which harboring singly the dCpf1 and the other one the co-transformed dCpf1 and Lambda-GFP-RFP constructs.
Week 18 (9th of September – 15th of September)
Final transformation of the Cpf1 spacers into the competent cells harboring dCpf1 and Lambda-GRP-RFP. Inhibition of GFP and RFP is a fact!
Week 19 (16th of September – 22nd of September)
Preforming plate reader experiments to assure we have proper inhibition of GFP and RFP under the transcription of the regulatory region of bacteriophage Lambda.
Week 20 (23rd of September – 29th of September)
Preforming plaque assays with Marijn for the inhibition of bacteriophage lambda with dCpf1 and the assessment for the best produced spacers. .
Week 21 (30th of September – 6th of October)
Acquired and transformed life Cpf1 with bacteriophage lambda targeting spacers into E. coli. Preforming a growth-curve analyses of both Cpf1 and dCpf1 in the plate reader to inhibit bacteriophage Lambda proliferation.
October
Week 22 (7th of October – 13th of October)
Designing and engineering a CRISPR-Cas counter selection system for our modified bacteriophage and synthesizing the required Cpf1 spacers.
Week 23 (14th of October – 20th of October)
Finalizing lab work with the counter selection, gathering the last results of additional experiments, preforming data analyses and starting of writing for the wiki.