Team:Wageningen UR/Notebook/ProteinEngineering
These experiments were performed by Linea Muhsal.
May
Week 1
Setting up the lab, preparing media, preparing electrocompetent cells, primer design etc.
Week 2
PCR of Cry3Aa from Bacillus thuringiensis var. tenebrionis and Cry1Ab from Bacillus thuringiensis Berliner 1915. Success with PCR of Cry3Aa, but not Cry1Ab. Creation of vector pBbA7c-Cry3Aa.
Week 3
I tried to transform the created vector pBbA7c-Cry3Aa into Lemo21. No success.
Week 4
A lot of time spent on trying to clone the vector pBbA7c-Cry3Aa into Lemo21. Inconclusive results.
June
Week 5-8
No chance of working in the lab due to moving of the lab.
July
Week 9
A fresh start with a lot of work for setting up the lab again. I learned the basics of phage work in this and the next week.
Week 10
Continuation of learning how to work with bacteriophages. Mite feeding experiments with fluorophores: Mites do take up fluorophores with their food! But only a small amount of them (~16 %).
Week 11
Feeding experiment with mealworms. Mealworms are easy to feed, easy to dissect and easy to keep. They take up fluorescein when fed with carrots dipped in it.
Week 12
vacation
August
Week 13
SDS-PAGE of mealworm proteins, mite proteins, and mealworm gut proteins.Proteins can be seen in all of the samples. There is a distinct difference in protein pattern in the gut and the overall mealworm sample. This indicates that using only the midgut for experiments enhances the chances of finding a specific toxin. Attempts to do so in mites failed due to the small size of Varroa destructor.
Week 14-15
The titre of the provided phage library was to be determined. Attempts to do so via "tear-drop assay", where one X-Gal/IPTG plate can be used to characterize 4 samples, failed. This is probably due to the high mobility of the phage M13KE. The titre was obtained by using the protocol listed here (LINK).
In vivo phage display on mites and mealworms has been performed. Titre dropped drastically after each round.
Week 16
In vivo phage display on mealworms with propagation of phages inbetween the rounds. During this, unexpected, lytic phages appeared on the titre plates. Experiments were put on hold.
September
Week 17
Investigation into the source of contamination. Phages appear to originate from the mealworms. In vivo phage display experiments were cancelled.
Week 18
Construction of Cry3Aa-expression plasmids with random binding site mutations. Transformation into Lemo21.
Week 19
In vitro phage binding display.
Week 20
Expression and first screening of Cry3Aa mutants.
October
Week 21
Final toxicity analysis of Cry3Aa mutants. Data analysis.
