Difference between revisions of "Team:Tuebingen/Labwork"

 
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{{Tuebingen/TitleQuote|quote=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.|person=David Cronenberg}}
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<html><img id="titleImage" src="https://static.igem.org/mediawiki/2018/0/09/T--Tuebingen--Placeholder.jpg" alt="Placeholder"></html>
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{{Tuebingen/SectionStart|id=Introduction|title=Introduction}}
 
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Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. 
 
  
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Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
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{{Tuebingen/PlainSubHeading|BoNT C - Liscense to enter}}
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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.
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{{Tuebingen/SingleContent|
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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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{{Tuebingen/SingleContent|
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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/SectionStart|id=Methods|title=Methods}}
 
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Here we're describing our methods. You can click any of the tabs to see what we did in each laboratory.
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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.
 
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<html><div class="tabMenu"><button onclick="openTab('MolbioMethods', 'tabMethods')">Molecular Biology</button><button onclick="openTab('ProteinsMethods', 'tabMethods')">Protein Expression</button><button onclick="openTab('CellsMethods', 'tabMethods')">Cell Culture</button><button onclick="openTab('ChemistryMethods', 'tabMethods')">Chemistry</button></div>
 
<html><div class="tabMenu"><button onclick="openTab('MolbioMethods', 'tabMethods')">Molecular Biology</button><button onclick="openTab('ProteinsMethods', 'tabMethods')">Protein Expression</button><button onclick="openTab('CellsMethods', 'tabMethods')">Cell Culture</button><button onclick="openTab('ChemistryMethods', 'tabMethods')">Chemistry</button></div>
 
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{{Tuebingen/Link|text=Electrocompetent Cells|url=https://static.igem.org/mediawiki/2018/4/46/T--Tuebingen--Electrocompetent_Cells.pdf}}
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Generating Electrocompetent Cells
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{{Tuebingen/Link|text=Electroporation|url=https://static.igem.org/mediawiki/2018/0/0c/T--Tuebingen--Electrocoporation.pdf}}
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Transformation of Cells
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{{Tuebingen/Link|text=Mini Prep|url=https://static.igem.org/mediawiki/2018/3/30/T--Tuebingen--MiniPrep.pdf}}
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Mini Preparation of Plasmid DNA
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{{Tuebingen/Link|text=Maxi Prep|url=https://static.igem.org/mediawiki/2018/9/92/T--Tuebingen--MaxiPrep.pdf}}
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Maxi Preparation of Plasmid DNA
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{{Tuebingen/Link|text=Restriction Digest and Ligation|url=https://static.igem.org/mediawiki/2018/a/a4/T--Tuebingen--RestrictionLigation.pdf}}
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Restriction Digest and Ligation
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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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{{Tuebingen/Link|text=Distillation|url=https://static.igem.org/mediawiki/2018/5/52/T--Tuebingen--Chemie_desti.pdf}}
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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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Explanation of chromatographie
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{{https://static.igem.org/mediawiki/2018/c/cc/T--Tuebingen--Chemie_Dry_Solvent.pdf|description=Drying of Solvents}}
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{{Tuebingen/Link|text=Drying|url=https://2018.igem.org/File:T--Tuebingen--Chemie_Dry_Solvent.pdf}}
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Explanation how to dry solvents
 
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Hier sollen dann die Beschreibungen der PDF's stehen
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{{Tuebingen/Link|text=OH to SH|url=https://static.igem.org/mediawiki/2018/9/95/T--Tuebingen--Chemie_OH-SHalspdf.pdf}}
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Synthesis of the Thiol-Esli
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{{Tuebingen/Link|text=SH to SS|url=https://static.igem.org/mediawiki/2018/b/b4/T--Tuebingen--Chemie_SHSS.pdf}}
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Synthesis of the Disulfid-Esli
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{{Tuebingen/Link|text=Azid-D|url=https://static.igem.org/mediawiki/2018/1/18/T--Tuebingen--Chemie_AzidD.pdf}}
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Synthesis of the Azid-Donor
 
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{{Tuebingen/SectionStart|id=Labbook|title=Labbook}}
 
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This is our Labbook. It is the place, where we describe everything that we did.
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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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<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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{{Tuebingen/PlainSubHeading|13.08.2018}}
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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 N2 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 produkt.
 
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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 Sulfurylchlorid.
 
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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/H2O, treated with Cysteamin 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, colourless 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, colourless solid.
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{{Tuebingen/SectionStart|id=Interlab|title=Interlab}}
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The S-S-Eslicarbazepine was further purified by washing with ethyl acetate and freeing of water.
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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.  
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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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The main question the InterLab Study 2018 tackeled was:
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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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{{Tuebingen/PlainSubHeading|29.08.2018}}
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{{Tuebingen/PlainSubHeading|Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?}}
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It was tried to purify the reactants for further use, but it gave no yield.
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If you are interested to read more about our InterLab study results, click here:}}
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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