Team:Wageningen UR/Notebook/Marijn
CELL-FREE TRANSCRIPTION-TRANSLATION
By: Marijn Ceelen
Marijns notebook discusses not only Cell-Free Expression Systems, used for phage engineering and protein production, but also more general phage engineering approaches. His results can be found here and here.
May
Week 1 (13th of May – 19th of May)
Finalizing the plan for the project and dividing the project in subprojects.
Week 2 (20th of May – 26th of May)
Divide the subprojects over team members.
Week 3 (27th of May – 2nd of June)
Start writing proposal. Literature research into TXTL systems and phage display.
June
Week 4 (3rd of June – 9th of June)
Write proposal. Looking into phage genome engineering.
Week 5 (10th of June – 16th of June)
Write proposal.
Week 6 (17th of June – 23rd of June)
Write proposal.
Week 7 (24th of June – 30th of June)
Start in the lab. Revive old phage stocks by infecting E. coli.
July
Week 8 (1st of July – 7th of July)
Start testing old phage stocks with plaque assays.
Week 9 (8th of July – 14th of July)
Make phage lambda stocks.
Week 10 (15th of July – 21st of July)
Genome isolation (isopropanol precipitation method) and test by digestion and PCR. No good results. PCR does not show bands and digestion does not show fragments.
Week 11 (22nd of July – 28th of July)
Repeat genome isolation and follow up with cleanup using Genejet genomic DNA kit. Repeat of digestion and PCR, still no result.
Week 12 (29th of July – 4th of August)
New genome isolation method using phenol and chloroform. PCR gives unexplainable bands. Digestion still does not show fragments.
August
Week 13 (5th of August – 11th of August)
Repeat chloroform phenol genome isolation, still no result.
Week 14 (12th of August – 18th of August)
First trial for TXTL phage lambda production. No phages detected.
Week 15 (19th of August – 25th of August)
Pulse field electrophoresis to visualize genome on gel. This gives a smear on the gel instead of the band at 48 kb that we expect. This indicates low quality of DNA.
Week 16 (26th of August – 1st of September)
Revive phage T7 stock. TXTL production of MAMPs for Cleo.
September
Week 17 (2nd of September – 8th of September)
Phenol chloroform genome isolation for phage T7. Digestion of genome shows good results. TXTL test of phage T7 and phage lambda production. T7 production successful.
Week 18 (9th of September – 15th of September)
Trying to solve lambda TXTL production using concatemers. Long stretches of ligated phage lambda genomes. Also repeat T7 phage production to quantify phage titer. Lambda production using concatemers not successful.
Week 19 (16th of September – 22nd of September)
Visualization of phage lambda and phage T7 using electron microscopy. Start cloning phage lambda Cro gene as addition to TXTL reaction. Some cloning problems because of toxicity of Cro protein at high expression.
Week 20 (23rd of September – 29th of September)
Cro cloning under weak Anderson promoter and T7 promoter.
Week 21 (30th of September – 6th of October)
Cro cloning confirmed successful by sequencing. Start phage lambda engineering using lambda red recombination system. In silico design hybrid phage T7 and lambda constructs. Cro TXTL trial.
October
Week 22 (7th of October – 13th of October)
Sequencing constructs for phage lambda engineering. Hybrid phage system cloning.
Week 23 (14th of October – 20th of October)
Production of recombinant phages using lambda red system and recombination cassette. Counterselection for recombinant phages using CRISPR-Cas. Screening of phages using plaque PCR and sequencing. Chitin binding assay on the engineered phages.