Team:Bielefeld-Germany/Labjournal

From 2013.igem.org

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==Overview October==
==Overview October==
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*Successful cultivations of ''Escherichia coli'' KRX with OprF and GldA in our Microbial Fuel Cell.
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** NADH overproduction for the GldA strain improves extracellular electron transfer mediated by NADH and resulting in an 20 % increased bioelectricity output.
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**''Escherichia coli'' KRX with OprF shows 100 % higher voltage and electric charge than ''E. coli Wildtyp''. Electron shuttle-mediated electron transfer across the membrane is greatly improved by heterologous expression of outer membrane porin OprF.

Revision as of 10:16, 3 October 2013



Journal



Milestones





Overview Prologue

  • The iGEM-Team Bielefeld 2013 started working and decided to deal with the project 'Microbial Fuel Cell'.


Overview May

  • Initiating labwork on the sub-project endogenous mediator Glycerol dehydrogenase.
  • Starting lab work with the first successful PCR's. The main work however is still in the planning of our project. Designing experiments and a lot of research.
  • Finding sponsors goes ahead. Many companies like our project and want to support us.


Overview June

  • Starting labwork on the sub-project Porins.
  • Successful PCR on OprF gene from Pseudomonas fluorescens. OprF with Pre- and Suffix overlaps could be amplified from genome.
  • Planning of our Human Practice projects started and the first participations are fixed.


Overview July

  • Poster presentation at the congress ‘[http://www.biotechnologie2020plus.de Next generation of biotechnological processes 2020+]’ in Berlin.
  • Sponsoring acquisition is nearly finished.
  • Problem with cloning delaying the progress in the laboratory.
  • We have published our [http://ekvv.uni-bielefeld.de/blog/uniaktuell/entry/mit_bakterien_batterie_strom_erzeugen first press release] with a great response. The press release was picked up worldwide.


Overview August

  • Our great expert Dr. Falk Harnisch has answered numerous questions about our project and helped us very well.
  • GldA BioBrick (<bbpart>BBa_K1172201</bbpart>) was examined.
    • GldA Biobrick Devices with different promotors and RBS could be added: (<bbpart>BBa_K1172203</bbpart>, <bbpart>BBa_K1172204</bbpart>, <bbpart>BBa_K1172205</bbpart>)
  • OprF BioBrick (<bbpart>BBa_K1172501</bbpart>) is available.
    • OprF Biobrick was functionalized with different promotors and RBS: (<bbpart>BBa_K1172502</bbpart>, <bbpart>BBa_K1172503</bbpart>, <bbpart>BBa_K1172504</bbpart>, <bbpart>BBa_K1172505</bbpart>, <bbpart>BBa_K1172507</bbpart>)
  • Successful protein expression and overproduction of glycerol dehydrogenase. SDS-PAGE shows GldA at expected size.


Overview September

  • Successful characterization of Glycerol dehydrogenase
    • NADH-Assays show an increasing intra- and extracellular NADH concentration for Escherichia coli KRX with overexpression of glycerol dehydrogenase.
    • Glycerol dehydrogenase was examined by MALDI-TOF MS/MS with a Mascot Score of 266 against Escherichia coli database.


  • Great characterization of outer membrane porin OprF
    • Successful Hexadecan-Assay for characterization of hydrophobicity of the outer membrane. The hydrophobicity increases continuously with increasing promoter strength up to 221% hydrophobizity in contrast to wildtyp strain.
    • ONPG and NPN uptake assays were performed. Membrane permeability is continuously increasing for Escherichia coli with heterologeous expression of OprF.
    • According to AFM images, E. coli KRX with OprF shows a slightly rougher cell surface morphology in contrast to Escherichia coli KRX Wildtyp, which confirms the results.
    • We can see a strong overexpression band on SDS-PAGE at the expected OprF size of about 36 kDa which is equated with a strong expression and overproduction of OprF. Furthermore outer membrane porin was examined by MALDI-TOF MS/MS with a Mascot Score of 222 against bacteria database.


  • We supported the CeBiTec student academy with supervising the ‘Synthetic Biology’ experiment and the day of synthetic biology in the city of Bielefeld.
  • Our team assigned the Track Food & Energy in this year, with the project title ‘Ecolectricity – currently available’.


Overview October

  • Successful cultivations of Escherichia coli KRX with OprF and GldA in our Microbial Fuel Cell.
    • NADH overproduction for the GldA strain improves extracellular electron transfer mediated by NADH and resulting in an 20 % increased bioelectricity output.
    • Escherichia coli KRX with OprF shows 100 % higher voltage and electric charge than E. coli Wildtyp. Electron shuttle-mediated electron transfer across the membrane is greatly improved by heterologous expression of outer membrane porin OprF.



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