Team:Saarland/Test

From 2014.igem.org

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<h1> Labbook </h1>
 
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<b> To Read More: Click on our Timeline </b>
 
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<img src="https://static.igem.org/mediawiki/2014/b/ba/Inhaltverzeichnis.png" width="900px";alt="Inhaltsverzeichnis"
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<iframe src="//player.vimeo.com/video/109005558" width="500" height="281" frameborder="0" webkitallowfullscreen mozallowfullscreen allowfullscreen></iframe> <p><a href="http://vimeo.com/109005558">Saarland University iGEM 2014 - Campus Tour</a> </p></html>
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<a id="February"> </a>
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<h3> February</h3>
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[[File: Mark ErlenmeyerKL.png|thumb|30px|left]]Develop strategy for the first ever biotechnological production of the naked mole rat´s high molecular mass hyaluronic acid and the characterisation of its inhibitory effects on carcinogenesis.
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*Recruiting committed team members.
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<h3> March </h3>
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*Looking for sponsors.
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*Team registration.
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<h3>April</h3>
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*Looking for sponsors
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*Design of a team logo
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<h3>May </h3>
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*Looking for sponsors
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*Conception and design of the team wiki
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[[File: Mark ErlenmeyerKL.png|thumb|30px|left]]
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Waiting for completion of <i>has</i>2 gene synthesis.
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<h4>Highlight at Week 4</h4>
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*Meet up of German iGEM teams in Munich. Inspiring Weekend. We enjoyed the short trip.
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<h3>June</h3>
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<h4>1. Week</h4>
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[[File: Mark ErlenmeyerKL.png|thumb|30px|left]]Finally, the synthesised <i>has</i>2 gene has arrived!
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[[File: Mark ErlenmeyerKL.png|thumb|30px|left]]
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Transformation of electro competent <i>E. coli</i> cells with Eurofins standard delivery plasmid pexK4 containing the synthesised <i>has</i>2 insert DNA.
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[[File: Mark ErlenmeyerKL.png|thumb|30px|left]]Plasmid preparation and control digestion to confirm the presence of <i>has</i>2 insert DNA.
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<h4>2. Week</h4>
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[[File: Mark ErlenmeyerKL.png|thumb|30px|left]]First attempt to insert <i>has</i>2 DNA into the multiple cloning site (MCS) of pSMF2.1 plasmid construct. This shuttle plasmid allows on the one hand the amplification of plasmid DNA in high copy numbers in <i>E. coli</i> cells. On the other hand pSMF2.1 is optimised for expression of recombinant proteins in our favourite chassis <i>B. megaterium. Has</i>2 insert DNA was absent in transformants.
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<h4>3. Week</h4>
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[[File: Mark ErlenmeyerKL.png|thumb|30px|left]]Repetition of cloning experiments (week 2) with new T4 ligase provided many <i>E. coli </i>transformants with pSMF2.1 plasmid construct containing <i>has</i>2 insert DNA.
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<h4>4. Week</h4>
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[[File: Mark ErlenmeyerKL.png|thumb|30px|left]]
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Extraction of genomic DNA from <i>B. megaterium</i> was performed.
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[[File: Mark ErlenmeyerKL.png|thumb|30px|left]]Optimal PCR conditions for the amplification of genomic DNA from <i>B. megaterium </i>were tested. First gene (<i>UDP-glc</i>DH) involved in synthesis of precursor molecules and probably contributing to a high yield production of high molecular mass hyaluronic acid was successfully amplified.
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[[File: Mark ErlenmeyerKL.png|thumb|30px|left]]
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First attempt for downstream insertion of <i>UDP-glc</i>DH DNA into the MCS of pSMF2.1 plasmid construct that already contained the <i>has</i>2 insert DNA was successful.
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<h3>July</h3>
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<h4>1. Week</h4>
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*Open day at Saarland University. The iGEM team was proud to present its project to the general public.
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[[File: Mark ErlenmeyerKL.png|thumb|30px|left]]Plasmid preparation and control digestion to confirm the simultaneous presence of <i>has</i>2 and <i>UDP-glc</i>DH insert DNA in the pSMF2.1 plasmid construct.
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[[File: Mark ErlenmeyerKL.png|thumb|30px|left]]Preparation of fresh protoplasts for transformation of <i>B. megaterium</i> strain MS941 was successful. Competence is outstanding.
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<h4>2. Week</h4>
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[[File: Mark ErlenmeyerKL.png|thumb|30px|left]]Transformations of <i>B. megaterium</i> protoplasts with different pSMF2.1 plasmid constructs were successful. Screening process for the identification of <i>B. megaterium</i> transformants with correct plasmid constructs was time consuming.
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[[File: Mark ErlenmeyerKL.png|thumb|30px|left]]<i>B. megaterium</i> chassis for the first ever biotechnological production of high molecular mass hyaluronic acid is ready!
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<h4>3. Week</h4>
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[[File: Mark ErlenmeyerKL.png|thumb|30px|left]]Cultivation of <i>B. megaterium</i> and induction of recombinant protein production were performed. Samples were prepared after an appropriate cultivation time to check and characterise recombinant protein expression levels of Has2 and UDP-GlcDH.
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[[File: Mark ErlenmeyerKL.png|thumb|30px|left]]These samples were also used for viscosity measurement since the outstanding water binding capacity of high molecular mass hyaluronic acid should increase the viscosity of the cultivation medium.
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<h4>4. Week</h4>
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[[File: Mark BioBrickKL.png|thumb|40px|left]] Illegal restriction sites within <i>has</i>2 and <i>UDP-glc</i>DH were changed via QC-PCR. Resulting biobrick DNAs were inserted into the standard biobrick plasmid construct pSB1C3.
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[[File: Mark ErlenmeyerKL.png|thumb|30px|left]]Measurement of viscosity was performed. There was no difference in viscosity of the culture medium and consequently no high molecular mass hyaluronic acid production detectable in the first attempt.
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<h3>August</h3>
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<h4>1. Week</h4>
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[[File: Mark ErlenmeyerKL.png|thumb|30px|left]]
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SDS-PAGE
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[[File:Mark StromausfallKL.png|thumb|40px|left]]<br>
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Power failure at whole Saarland University during weekend. Reorder of heat sensitive enzymes including polymerases, restriction enzymes and ligase.
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[[File:Mark SNES-Controller.png|thumb|50px|left]]<br>
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Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project
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<h4>2. Week</h4>
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Functional enzymes arrived.
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[[File: Mark ErlenmeyerKL.png|thumb|30px|left]]Repetition of all <i>B. megaterium</i> transformations during timeout since corresponding glycerol stocks were damaged.
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[[File:Mark SNES-Controller.png|thumb|50px|left]]<br>
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Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project
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<h4>3. Week</h4>
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[[File:Mark eGFPKL.png|thumb|40px|left]]Generation of <i>has2-gfp</i> fusion gene via SOE-PCR for quick evaluation of Has2 expression level and localisation in <i>B. megaterium</i> was performed. Insertion of <i>has2-gfp</i> fusion gene into MCS of pSMF2.1 plasmid construct was successful. A lot of positive <i>E. coli</i> transformants!
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[[File:Mark SNES-Controller.png|thumb|50px|left]]<br>
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Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project
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<h4>4. Week</h4>
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[[File:Mark eGFPKL.png|thumb|40px|left]]Plasmid preparation and transformation of fresh prepared <i>B. megaterium</i> protoplasts with pSMF2.1 plasmid construct containing the <i>has2-gfp</i> fusion gene was successful.<br>
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[[File:Mark eGFPKL.png|thumb|40px|left]]Expression of Has-GFP fusion protein and samples for localisation studies via fluorescence microscopy were prepared.
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[[File:Mark SNES-Controller.png|thumb|50px|left]]<br>
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Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project
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<h3>September</h3>
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<h4>1. Week</h4>
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*Meet and greet with our sponsors. Thank you very much for your great support!
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[[File:Mark eGFPKL.png|thumb|40px|left]]First attempt to confirm Has-GFP expression via epi fluorescence microscopy was successful. <i>B. megaterium</i> cells glew beautifully green.
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[[File: Mark BioBrickKL.png|thumb|40px|left]]Additional genes involved in synthesis of precursor molecules were successfully amplified from genomic DNA of <i>B. megaterium</i> and inserted into the standard biobrick plasmid construct pSB1C3.
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[[File:Mark SNES-Controller.png|thumb|50px|left]]<br>
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Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project.
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* Evaluation of results and creation of figures for our team wiki.
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<h4>2. Week</h4>
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[[File:Mark eGFPKL.png|thumb|40px|left]]Expression of Has-GFP fusion protein was repeated and samples for localisation studies via laser scanning microscopy were prepared. No fluorescence signal was detectable after cultivation under the same conditions.
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[[File: Mark BioBrickKL.png|thumb|40px|left]]<br>
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Illegal restriction sites within the genes for pathway engineering were changed via QC-PCR.
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[[File:Mark SNES-Controller.png|thumb|50px|left]]<br>
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Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project.
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*Evaluation of results and creation of figures for our team wiki.
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<h4>3. Week</h4>
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[[File:Mark eGFPKL.png|thumb|40px|left]]Expression of Has-GFP fusion protein was tested under different cultivation conditions. No significant fluorescence signal. Possible misfolding of Has-GFP protein
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Sequencing of biobricks was prepared.
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Western blot of Has-GFP to determine the presence of misfolded protein.
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Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project.
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Waiting for sequencing results.
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Human Practices: Design and development of the roleplaying game for the ethical and social implications of our project.
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*Evaluation of results and creation of figures for our team wiki.
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<h3>October</h3>
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<h4>1. Week</h4>
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Biobrick sequencing was finished. Biobricks are ready to ship to the iGEM Headquarter.
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Human Practices: Beta test of the roleplaying game for the ethical and social implications of our project.
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*Evaluation of results and creation of figures for our team wiki.
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<h4>2. Week</h4>
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*Last modifications on team wiki!
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[[File:Mark SNES-Controller.png|thumb|50px|left]]<br>
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Human Practices: Last modifications of the roleplaying game for the ethical and social implications of our project.
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{{Team:Saarland/footer}}
{{Team:Saarland/footer}}

Latest revision as of 12:39, 17 October 2014


Retrieved from "http://2014.igem.org/Team:Saarland/Test"