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Expression of the protein and harvesting of the bacterial culture
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Transformation of Cells
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Mini Preparation of Plasmid DNA
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Revision as of 21:59, 17 October 2018

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
Title image
Introduction

BoNT C - Liscense to enter

As stated, we modify botulinum toxin in a way that leads to its detoxification. This, and the coupling to other substances was the main part of our Labwork, next to cell culture to test our library on neuron-like cells. The whole process that took place and different methods we used to archive our goals is described below.
An other important part of every iGEM year is the InterLab Study, which is one of the Bronze-Medal Criteria.
If you are intersted in our Bioinformatic part: BioInfo


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.

Electrocompetent Cells
Generating Electrocompetent Cells
Electroporation
Transformation of Cells
Mini Prep
Mini Preparation of Plasmid DNA
Maxi Prep
Maxi Preparation of Plasmid DNA
Restriction Digest and Ligation
Restriction Digest and Ligation


Expression and harvesting
Expression of the protein and harvesting of the bacterial culture
Electroporation
Transformation of Cells
Mini Prep
Mini Preparation of Plasmid DNA
Maxi Prep
Maxi Preparation of Plasmid DNA
Restriction Digest and Ligation
Restriction Digest and Ligation


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Distillation
Explanation of distillation
Chromatographie
Explanation of chromatographie
Drying
Explanation how to dry solvents
OH to SH
Synthesis of the Thiol-Esli
SH to SS
Synthesis of the Disulfid-Esli
Azid-D
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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This is a text
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13.08.2018

Beginning of the synthesis of the Thiol-Eslicarbazepin in Toluen. Eslicarbazepin 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 CaH2 overnight.

15.08.2018

Distillation of 1,2-dimethoxyethane, treating with N2 for inert storage.

16.08.2018

Alternative synthesis of the Thiol-Eslicarbazepin 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 Thiol-Eslicarbazepin and purification by column chromatography (-> methods). A sample was taken and given to mass spectrometry.

20.08.2018

The MS-Data was examined showed indications of large impurities. Further purification of the Thiol-Eslicarbazepin by column chromatography.

21.08.2018

First attempt of the synthesis of the azide-Donor (method Azid-D). Sodium 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 showed no yield. The second attempt for the synthesis of the azide-donor was started. Sulfuryl chloride was treated with sodium azide 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. No product was obtained. The third attempt for the synthesis of the azide-donor was started. An ice-cooled Suspension of sodium azide in acetonitrile was treated with Sulfuryl chloride. Synthesis Disulfide-Eslicarbazepine was started. The synthesis was performed under inert conditions. The Thiol-Eslicarbazepin was solved in THF/H2O, treated with Cysteamine and stirred overnight.

24.08.2018

The ice-cooled solution of the azide was treated portion-wise with Imidazole. It was stirred for 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 in a rotary evaporator to obtain a crystalline, colorless solid.

27.08.2018

The S-S-Eslicarbazepine was further purified by washing with ethyl acetate. A sample of the S-S-Eslicarbazepine was taken and given to mass spectrometry.

28.08.2018

The MS-Data was examined. MS-Data suggested the reaction was not carried out as the original reactants were reobtained.

29.08.2018

The reactants were dried with a rotary evaporator for further use, but Thio-Eslicarbazepine was destroyed due to it's instability in water.

30.08.2018

Destroying of the azide-donor.

31.08.2018

Cleaning


Interlab

In this year, our team again 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:

Dies ist ein eine Beschreibung