ToxR Signal Transduction

By: Tom van der Aalst

Here, the work of Tom on the ToxR dimerization system is described. Unfortunately, things didn't really work out for him because he needed more time for his project, which is why it sadly could not be incorporated into Mantis.

May

Week 1 (1st of May- 7th of May)
Getting started, proposal writing, office decoration.

Week 2 (8th of May- 14th of May)
Proposal writing, assess iGEM 2016 inventory and leftovers. Assume duty as Lab Manager.

Week 3 (15th of May – 21st of May)
Experiment design, John van der Oost meeting, literature search.

Week 4 (22nd of May – 28th of May)
Further experiment planning. Deriving sequences of parts from UniProt and BLAST searches, among others.

June

Week 5 (29th of May – 4th of June)
Modify experimental setup. Continued looking for sequences. Talked about patents.

Week 6 (5th of June – 11th of June)
Generating constructs in silico, established fragment origins and noted suitable BioBricks.

Week 7 (12th of June – 18th of June)
Finished and sent proposal. Played around some more with SnapGene.

Week 8 (18th of June – 24th of June)
Decided on final experiment form, designed constructs with SnapGene around this and prepared primers.

July

Week 9 (25th of June - 1st of July)
Started labwork by preparing antibiotics, LB media, agar plates, etc. Designed and ordered my gBlock.

Week 10 (3rd of July - 9th of July)
Received gBlock and primers, diluted them to working stocks and storage stocks. Poured extra LB agar plates with Tet and Tet+Cam. Went to Delft European meetup.

Week 11 (10th of July - 16th of July)
Started spheroplast viability assay. Amplified parts of the gBlock by PCR for future use in vector cloning.

Week 12 (17th of July - 23th of July)
Continued (this time successful) PCR amplification of the ToxR and IgG Affibody domains from the geneBlock. Designed primers for isolation of Maltose-Binding Protein from a donated plasmid.

Week 13 (24th of July - 30th of July)
Secured FM4-64 dye for membrane staining of spheroplasts and performed protocol for creating those. Performed PCR for fragments used in later constructs and successfully isolated those. Digested all parts for construct 1 so far and gel purified the adequate bands.

August

Week 14 (31st of July - 6th of August)
Transformed cells with construct 1 and created glycerol stocks from those. Prepared a protocol for the generation of giant spheroplasts and executed it, but to no avail so far.

Week 15 (7th of August - 13th of August)
Sent construct 1 for sequencing but was disappointed to see that assembly did not work. Repeated assembly and performed corresponding colony PCR, sent samples for sequencing again. Assessed whether Q5 Mastermix from last year’s team works or not (it did, aside from 1 Epp).

Week 16 (14th of August - 20th of August)
Started generation of the ctx promoter by PCR of annealed oligos, as proposed by Marta and me. Looked good on gel. Attempted another assembly of Construct 1, but sequencing results have proven yet again that it didn’t work.

Week 17 (21st of August - 27st of August)
Made preparations for the assembly of the visualization plasmid and performed it as well. No results. Tried HiFi assembly mix from NEB and performed cPCR the day after for Construct 1. Still nothing.

September

Week 18 (28th of August - 3rd of September)
Decided to go for full Golden Gate assembly, ordered new primers and performed new PCRs for the generation of the correct overhangs. Had some trouble with amplification of the pSB3T5 backbone but managed to solve it.

Week 19 (4th of September - 10th of September)
Performed multiple attempts at Golden Gate assembly, discovered that BbsI is not functioning correctly after a separate assessment. Made electrocompetent cells for high(er) efficiency transformations in the future.

Week 20 (11th of September - 16th of September)
Used newly ordered BbsI-HF for new assembly reactions after assessing its activity. Still refused to work due to reasons unknown.

Week 21 (17th of September - 23rd of September)
Played around with reaction conditions (buffer composition, incubation times, etc.) to see if it enhances results. Colony PCRs showed that it has little to no effect. Ordered new primers again using another TypeIIS enzyme (BsmBI), since BbsI-HF comes in unpractical volumes and is quite expensive, compared to other enzymes.

Week 22 (September 24th - 30th of September)
pSB3T5 amplification giving problems again, designed primers to isolate this backbone using a 2-step assembly. PCR’ed like a madman, no luck so far.

October

Week 23 (October 1st - October 7th)
One last shot of continuing the project, otherwise it will be dropped and I will be assigned to help Natalia, among others. Also involved in the side project of drying bacteria now to contribute some more to the total project.

Week 24 (October 8th - October 14th)
Project dropped, sadly. My remaining time will be divided over subjects that require help. Will still try to finish my own constructs, but with heavily decreased priority. Planning to perform drying experiments for now. Miraculously finished my first construct somehow.

Week 25 (October 15th - October 21st)
More drying experiments. For my own project, pSB3T5 amplification is giving problems again, unfortunately. Despite these setbacks, managed to finish yet another construct.

Week 26 (October 22nd - October 28th)
PCR of pSB3T5 still doesn’t want to work, despite using the exact same conditions which did the trick last time and slight variations upon that.

Team:Wageningen UR/Notebook/Journal/ToxR