Team:Tuebingen/Labwork
Labwork
Everybody's a mad scientist, and life is their lab. We're all trying to experiment to find a way to live, to solve problems, to fend off madness and chaos.- David Cronenberg
Introduction
BoNT C - Licence to enter
In modern medicine treatment options involve many substances modified from natural sources, occasionally even toxins.
We modify botulinum toxin in a way that leads to its detoxification. Thus, it can be coupled with a variety of other substances while not losing its specific shuttle mechanism for neuronal cells. In detail, we develop a library of different detoxified botulinum toxin derivatives which can accommodate other proteins, small molecules, and fluorochromes by specific linkers.
To investigate the influence of the point mutations leading to detoxification in the active site, we conduct MD simulations. Since our shuttle mechanism could potentially be used in patients, we remove the most prevalent immune epitopes by a theoretical bioinformatics approach.
Ultimately, our system is supposed to be utilized for therapy strategies and specific neuronal targeting in basic research.
With our project we want to encourage future teams to think outside the box while keeping safety in mind.
Methods
Working neatly is very important to every scientist. Because of this, here we're describing our methods. You can click any of the tabs to see what we did in each laboratory.
Generating Electrocompetent Cells
Transformation of Cells
Mini Preparation of Plasmid DNA
Maxi Preparation of Plasmid DNA
Restriction Digest and Ligation
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Explanation of distillation
Explanation of chromatographie
Explanation how to dry solvents
Synthesis of the Thiol-Esli
Synthesis of the Disulfid-Esli
Synthesis of the Azid-Donor
Labbook
Here you can read our Labbook. It is the place, where we described everything that we did, and when. If you are interessted in our workflow, you can read everything below.
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13.08.2018
Beginning of the synthesis of the Thiol-Eslicarbazepin in Toluen.
Esli-OH was treated with 0.5 mole equivalent of Lawesson's reagent in Toluene.
Reflux at 120°C gave a yellow liquid with white solids.
Purification with HPLC.
A sample was taken and given to mass spectrometry.
14.08.2018
The MS-Data was examined.
MS-Data gave a yield of >10%.
Drying of 1,2-dimethoxyethane with CuH overnight.
15.08.2018
Distillation of 1,2-dimethoxyethane, treating with N2 for storing inert.
16.08.2018
Alternative synthesis of the Esli-SH treated with 1,2-dimethoxyethane (DME) at room temperature with 0.5 mole equivalent of Lawesson's reagent, (method OH->SH).
17.08.2018
Drying the Esli-SH and purification by column chromatography (-> methods).
A sample was taken and given to mass spectrometry.
20.08.2018
The MS-Data was examined.
It gave indications of large impurities.
Further purification of the Esli-SH by column chromatography.
21.08.2018
First attempt of the synthesis of the Azid-Donor (method Azid-D). Natrium-Azide was suspended in Sulfuryl chloride and stirred overnight.
22.08.2018
The azide mixture was treated with Imidazole and dried in vacuo. After suspending in ethyl acetate the solution was treated with sulfuric acid to yield a crude, yellow solid.
A sample was taken and given to mass spectrometry.
23.08.2018
The MS-Data was examined.
MS-Data gave a no yield.
The second attempt for the synthesis of the Azid-Donor was started.
Sulfuryl chloride was treated with Natriumazid and imidazole, suspended in Acetonitrile and stirred for three hours.
The product was dried in vacuo, solved in ethyl acetate and treated with sulfuric acid to give no product.
The third attempt for the synthesis of the Azid-Donor was started.
An ice-cooled Suspension of Natriumazid in Acetonitrile was treated with Sulfuryl chloride.
Synthesis Disulfide-Eslicarbazepine was started.
The synthesis was performed under inert conditions. The SH-Esli was solved in THF/H2O, treated with Cysteamine and stirred overnight.
24.08.2018
The ice-cooled solution of the Azid was portion-wise treated with Imidazole.
It was stirred or three hours, diluted with ethyl acetate, washed, saturated and treated with sulfuric acid.
After stirring for one hour the crude, colorless product was obtained.
The product was dried in vacuo.
The solution of the S-S-Eslicarbazepine was freed of the solvent to obtain a crystalline, colorless solid.
27.08.2018
The S-S-Eslicarbazepine was further purified by washing with ethyl acetate and freeing of water.
A sample of the S-S-Eslicarbazepine was taken and given to mass spectrometry.
28.08.2018
The MS-Data was examined.
MS-Data gave a no yield, the reaction was not carried out as the pure reactants were obtained.
29.08.2018
It was tried to purify the reactants for further use, but it gave no yield.
30.08.2018
Destroying of the Azid-Donor.
31.08.2018
Cleaning
Interlab
Also in this year, our team decided to participate in the fifth international iGEM InterLab study, which aims to identify and correct the sources of systematic variability in synthetic biology measurements. The overall goal is that eventually, measurements taken in different labs will not underly the problems of variability due to different measurement environments or devices anymore, but will be reliable and comparable for all members of the science community.
The main question the InterLab Study 2018 tackeled was:
Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?
If you are interested to read more about our InterLab study results: click here.
