Difference between revisions of "Team:Tuebingen/Labwork"

 
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In modern medicine treatment options involve many substances modified from natural sources, occasionally even toxins.
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As stated, we modified botulinum toxin in a way that lead to its detoxification.  
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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.
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This, as well as the coupling of our detoxified BoNT C to other substances were the main aspects of our lab work, next to our special cell culture to test our library on neuron-like cells. The whole process we went through and the different methods we used to achieve our goals are described below.  
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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.
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Ultimately, our system is supposed to be utilized for therapeutic strategies and specific neuronal targeting in basic research.
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With our project, we want to encourage future teams to think outside the box while keeping safety in mind.
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An other important part of every iGEM year is the InterLab Study, which is one of the Bronze-Medal Criteria.
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If you are intersted in our Bioinformatic part: {{Tuebingen/Link|text=BioInfo|url=https://2018.igem.org/Team:Tuebingen/Bioinformatics}}
 
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{{Tuebingen/Link|text=Expression and harvesting|url=https://static.igem.org/mediawiki/2018/e/e6/T--Tuebingen--Expression_of_the_protein_and_harvesting_of_the_bacterial_culture.pdf}}
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Expression of the protein and harvesting of the bacterial culture
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{{Tuebingen/Link|text=His-tag purification|url=https://static.igem.org/mediawiki/2018/4/41/T--Tuebingen--His-tag_protein_purification.pdf}}
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His-tag protein purification
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{{Tuebingen/Link|text=Strep-tag purification|url=https://static.igem.org/mediawiki/2018/5/53/T--Tuebingen--Strep-tag_protein_purification.pdf}}
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Strep-tag protein purification
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{{Tuebingen/Link|text=Buffer preparation|url=https://static.igem.org/mediawiki/2018/a/ad/T--Tuebingen--Buffer_preparation.pdf}}
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Set up buffer
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{{Tuebingen/Link|text=Desalting samples|url=https://static.igem.org/mediawiki/2018/c/c8/T--Tuebingen--Desalting_samples.pdf}}
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Desalting samples
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{{Tuebingen/Link|text=SDS gel preservation|url=https://static.igem.org/mediawiki/2018/6/67/T--Tuebingen--SDS_gel_conservation.pdf}}
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SDS gel preservation
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{{Tuebingen/Link|text=Western blot|url=https://static.igem.org/mediawiki/2018/0/09/T--Tuebingen--Western_blot.pdf}}
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Western blot
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{{Tuebingen/Link|text=Basic cell culture|url=https://static.igem.org/mediawiki/2018/4/42/T--Tuebingen--BasicCellCulture.pdf}}
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SHSY5Y basic cell culture
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{{Tuebingen/Link|text=Freezing and thawing|url=https://static.igem.org/mediawiki/2018/d/d6/T--Tuebingen--FreezingThawing.pdf}}
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SHSY5Y freezing and thawing
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{{Tuebingen/Link|text=Cell splitting|url=https://static.igem.org/mediawiki/2018/c/c6/T--Tuebingen--CellSplitting.pdf}}
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SHSY5y cell splitting
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{{Tuebingen/Link|text=Differentiation|url=https://static.igem.org/mediawiki/2018/1/1d/T--Tuebingen--DifferentiationProtocol.pdf}}
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Differentiation protocol
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Explanation of distillation
 
Explanation of distillation
 
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{{Tuebingen/Link|text=Chromatographie|url=https://static.igem.org/mediawiki/2018/7/77/T--Tuebingen--Chemie_chroma.pdf}}
 
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Synthesis of the Azid-Donor
 
Synthesis of the Azid-Donor
 
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<html><div class="tabMenu"><button onclick="openTab('MolbioLB', 'tabLB')">Molecular Biology</button><button onclick="openTab('ProteinsLB', 'tabLB')">Protein Expression</button><button onclick="openTab('CellsLB', 'tabLB')">Cell Culture</button><button onclick="openTab('ChemistryLB', 'tabLB')">Chemistry</button></div><div id="tabLB" class="tabContent"></html>
 
<html><div class="tabMenu"><button onclick="openTab('MolbioLB', 'tabLB')">Molecular Biology</button><button onclick="openTab('ProteinsLB', 'tabLB')">Protein Expression</button><button onclick="openTab('CellsLB', 'tabLB')">Cell Culture</button><button onclick="openTab('ChemistryLB', 'tabLB')">Chemistry</button></div><div id="tabLB" class="tabContent"></html>
 
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{{Tuebingen/Link|text=MolBio Labbook|url=https://static.igem.org/mediawiki/2018/3/3e/T--Tuebingen--Labbook_Cloning.pdf}}
 
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Everything done in the MolBio Lab
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Beginning of the synthesis of the Thiol-Eslicarbazepin in Toluen.
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{{Tuebingen/Link|text=Purification, expression, toxassay|url=https://static.igem.org/mediawiki/2018/2/22/T--Tuebingen--Labbook_protein_expression.pdf}}
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Esli-OH was treated with 0.5 mole equivalent of Lawesson's reagent in Toluene.
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Reflux at 120°C gave a yellow liquid with white solids.
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Purification with HPLC.  
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A sample was taken and given to mass spectrometry.
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Labbook: protein purification, expression and toxassay
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The MS-Data was examined.
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MS-Data gave a yield of >10%.
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Drying of 1,2-dimethoxyethane with CuH overnight.
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Distillation of 1,2-dimethoxyethane, treating with N{{Tuebingen/Sub|2}} for storing inert.
 
 
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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).
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{{Tuebingen/Link|text=Cell Culture Labbook|url=https://2018.igem.org/File:T--Tuebingen--LabbookCellculture.pdf}}
 
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Labbook: Cell Culture
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Drying the Esli-SH and purification by column chromatography (-> methods).
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A sample was taken and given to mass spectrometry.
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The MS-Data was examined.
 
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It gave indications of large impurities.
 
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Further purification of the Esli-SH by column chromatography.
 
 
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First attempt of the synthesis of the Azid-Donor (method Azid-D). Natrium-Azide was suspended in Sulfuryl chloride and stirred overnight.
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{{Tuebingen/Link|text=Organic Chemistry Labbook |url=https://static.igem.org/mediawiki/2018/4/40/T--Tuebingen--Chemistry_Labbook.pdf}}
 
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Labbook: Organic synthesis
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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.
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A sample was taken and given to mass spectrometry.
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The MS-Data was examined.
 
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MS-Data gave a no yield.
 
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The second attempt for the synthesis of the Azid-Donor was started.
 
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Sulfuryl chloride was treated with Natriumazid and imidazole, suspended in Acetonitrile and stirred for three hours.
 
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The product was dried in vacuo, solved in ethyl acetate and treated with sulfuric acid to give no product.
 
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The third attempt for the synthesis of the Azid-Donor was started.
 
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An ice-cooled Suspension of Natriumazid in Acetonitrile was treated with Sulfuryl chloride.
 
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Synthesis Disulfide-Eslicarbazepine was started.
 
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The synthesis was performed under inert conditions. The SH-Esli was solved in THF/H{{Tuebingen/Sub|2}}O, treated with Cysteamine and stirred overnight.
 
 
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The ice-cooled solution of the Azid was portion-wise treated with Imidazole.
 
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It was stirred or three hours, diluted with ethyl acetate, washed, saturated and treated with sulfuric acid.
 
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After stirring for one hour the crude, colorless product was obtained.
 
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The product was dried in vacuo.
 
  
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The solution of the S-S-Eslicarbazepine was freed of the solvent to obtain a crystalline, colorless solid.
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The S-S-Eslicarbazepine was further purified by washing with ethyl acetate and freeing of water.
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A sample of the S-S-Eslicarbazepine was taken and given to mass spectrometry.
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The MS-Data was examined.
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MS-Data gave a no yield, the reaction was not carried out as the pure reactants were obtained.
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It was tried to purify the reactants for further use, but it gave no yield.
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Destroying of the Azid-Donor.
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Cleaning
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Latest revision as of 03:12, 18 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 modified botulinum toxin in a way that lead to its detoxification. This, as well as the coupling of our detoxified BoNT C to other substances were the main aspects of our lab work, next to our special cell culture to test our library on neuron-like cells. The whole process we went through and the different methods we used to achieve our goals are 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
His-tag purification
His-tag protein purification
Strep-tag purification
Strep-tag protein purification
Buffer preparation
Set up buffer
Desalting samples
Desalting samples
SDS gel preservation
SDS gel preservation
Western blot
Western blot
Basic cell culture
SHSY5Y basic cell culture
Freezing and thawing
SHSY5Y freezing and thawing
Cell splitting
SHSY5y cell splitting
Differentiation
Differentiation protocol
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.
MolBio Labbook
Everything done in the MolBio Lab
Purification, expression, toxassay
Labbook: protein purification, expression and toxassay
Cell Culture Labbook
Labbook: Cell Culture
Organic Chemistry Labbook
Labbook: Organic synthesis



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