Team:Bielefeld-Germany/Labjournal

From 2011.igem.org

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Molecular Beacons:
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[[https://2011.igem.org/Team:Bielefeld-Germany#NAD.2B_detection | Molecular Beacons]]:
* cloning the NAD+ dependent ligase from ''E. coli'' into BioBrick backbones
* cloning the NAD+ dependent ligase from ''E. coli'' into BioBrick backbones

Revision as of 12:27, 9 August 2011

On this page we summarize the (successful) results and achievements of our teamwork.

Contents

Week 1: 2nd - 8th may

Bisphenol A:

  • cloning of <partinfo>K123000</partinfo> and <partinfo>K123001</partinfo> behind weak (<partinfo>J23103</partinfo>) and medium strong (<partinfo>J23110</partinfo>) constitutive promoter (each part and both parts polycistronic)
  • cloning of fusionprotein between <partinfo>K123000</partinfo> and <partinfo>K123001</partinfo>, also assembly behind weak and medium strong constitutive promoter
  • testing and establishing of HPLC method for [http://en.wikipedia.org/wiki/Bisphenol_A BPA] detection
  • expression of the successfully assembled BPA degrading BioBricks in E. coli [http://openwetware.org/wiki/E._coli_genotypes#TOP10_.28Invitrogen.29 TOP10]

S-layer:

  • successful PCR on the S-layer genes of [http://expasy.org/sprot/hamap/CORGL.html Brevibacterium flavum] and Corynebacterium crenatum
  • successful cloning of the S-layer gene of B. flavum without TAT-sequence

Organizational:

  • meeting with [http://www.bielefeld-marketing.de/de/index.html Bielefeld Marketing] to discuss our participation at the science festival [http://www.geniale-bielefeld.de/ GENIALE] in Bielefeld

Week 2: 9th - 15th may

Figure 1: HPLC results of the first experiment on BPA degradation in E. coli TOP10. Cultivations were carried out in LB medium with 100 mg L-1 BPA at 30 °C. Samples were taken at the beginning of the cultivation and after one day. The HPLC results are shown above (area of the BPA peak). BisdA + BisdB is the polycistronic gene and BisdABisdB is the fusion protein.

Bisphenol A:

  • assembly of <partinfo>K157011</partinfo> behind existing BPA degrading parts (for purification and testing of <partinfo>K123000</partinfo> and <partinfo>K123001</partinfo> in a cell free system)
  • HPLC results: fusionprotein between <partinfo>K123000</partinfo> and <partinfo>K123001</partinfo> can degrade BPA and seems to work better than the polycistronic version (compare fig. 1)

S-layer:

  • expression of the S-layer gene without TAT-sequence of B. flavum in E. coli [http://www.promega.com/products/cloning-and-dna-markers/cloning-tools-and-competent-cells/bacterial-strains-and-competent-cells/single-step-_krx_-competent-cells/ KRX] and [http://openwetware.org/wiki/E._coli_genotypes#BL21.28DE3.29 BL21-Gold(DE3)] after sequencing gave correct results
  • successful PCR on the S-layer gene of [http://ijs.sgmjournals.org/cgi/content/abstract/54/3/779 Corynebacterium halotolerans]

Organizational:

  • moving to our own room in the [http://www.cebitec.uni-bielefeld.de/ CeBiTec]


Week 3: 16th - 22nd may

Bisphenol A:

  • successful PCR on the [http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.18.1.2 NADP oxidoreductase] gene from E. coli TOP10

S-layer:

  • successful cloning of the complete S-layer gene cspB of B. flavum, C. crenatum and C. halotolerans
  • successful cloning of the S-layer genes of B. flavum and C. halotolerans without TAT-sequence, without lipid anchor and without both (only self-assembly domain)

Organizational:


Week 4: 23rd - 29th may

Bisphenol A:

  • establishing a new method for analysis of BPA concentrations (extraction + LC-ESI-QTOF-MS)

S-layer:


Organizational:

  • arrange a BBQ for our workgroup in the CeBiTec to get to know our coworkers
  • substantiating our contribution to the GENIALE


Week 5: 30th may - 5th june

Bisphenol A:

  • beginning of first characterization experiments for BPA degrading BioBricks (<partinfo>K123000</partinfo> and <partinfo>K123001</partinfo>)

S-layer:


Organizational:

  • meeting with Prof. [http://de.wikipedia.org/wiki/Alfred_Pühler Alfred Pühler] to plan our contribution to the [http://www.cebitec.uni-bielefeld.de/content/view/209/88/ CeBiTec symposium] in july


Week 6: 6th - 12th june

Organizational:

  • presentation of the iGEM competition at the [http://www.bio.nrw.de/studentconvention 2nd BIO.NRW (PhD) Student Convention]


Week 7: 13th - 19th june

Bisphenol A:

  • cloning of NADP oxidoreductase in [http://www.bioinfo.pte.hu/f2/pict_f2/pJETmap.pdf pJET1.2] finally successful -> waiting for sequencing results to remove illegal restriction sites


Week 8: 20th - 26th june

Organizational:

  • finishing our first press release
  • all devices (thermocycler etc.) and materials (competent cells, polymerase, kits) from our sponsors arrived


Week 9: 27th june - 3rd july

S-Layer:

  • our synthesized S-Layers SgsE and SbpA finaly arrived


Week 10: 4th july - 10th july

Bisphenol A:

  • analyzing the influence of temperature, promotor intensity and the characteristics of the fusion protein on BPA degradation

Organizational:

  • presenting our posters from the teams of 2010 and 2011 at the congress [http://www.biotechnologie2020plus.de/BIO2020/Navigation/DE/root,did=121262.html Biotechnologie2020+] in Berlin hosted by the "Bundesministerium für Bildung und Forschung" (Federal Ministry of Education and Research).


Week 11: 11th july - 17th july

Bisphenol A / S-Layer:

  • our BioBrick order (some fluorescences proteins and cleavage sites) from iGEM HQ arrived

Organizational:

  • presenting the iGEM competition and our team project at the secondary school [http://www.rg-herford.de/ Ravensberger Gynmasium] in Herford ([http://www.flickr.com/photos/igem-bielefeld/sets/72157627214780020/ Photos])


Week 12: 18th july - 24th july

Organizational:

  • presenting our projects from 2010 and 2011 at the [http://www.cebitec.uni-bielefeld.de/content/view/209/88/ CeBiTec Symposium 2011] in Bielefeld
  • meeting and having fun with the iGEM teams from Delft/NL, Edinburgh/UK, Odense/DK. Freiburg/DE and Ljubljana/SL at the Symposium ([http://www.flickr.com/photos/igem-bielefeld/sets/72157627300144576/ Photos])


Bisphenol A / S-Layer:

  • removing illegal restiction sites to get valid BioBricks


[| Molecular Beacons]:

  • cloning the NAD+ dependent ligase from E. coli into BioBrick backbones

Week 13: 25th july - 31th july

S-Layer:

  • fusing the synthesized S-Layers to a bunch of fluorescent proteins

Bisphenol A:

  • testing new BPA extraction protocols for LC-MS including an internal standard (bisphenol F)

Week 14: 1st august - 7th august

Bisphenol A:

  • BPA analysis with extraction and LC-MS finally works and is very accurate
  • Better results for BPA degradation in E. coli -> our fusion protein (<partinfo>K123000</partinfo> to <partinfo>K123001</partinfo>) can completely degrade BPA
  • Measuring characterization results for different BPA degrading BioBricks

Molecular Beacons:

  • Successful characterization of two differently labeled Molecular Beacons as a preparation for the NAD+ bioassay including autonomously produced NAD+ dependent DNA ligase from E. coli


Week 14: 8th august - 14th august

Figure 1: HPLC results and optical density of experiments on BPA degradation of E. coli KRX. Cultivations were carried out in LB medium with ~ 100 mg L-1 BPA at 30 °C. Samples were taken every three hours over one day. BPA concentration was measured by HPLC with either UV detector or ESI-qTOF-MS. 502: negative control (<partinfo>K123000</partinfo>), 512: polycistronic <partinfo>K123001</partinfo> and <partinfo>K123000</partinfo>, 517: fusionprotein between <partinfo>K123001</partinfo> and <partinfo>K123000</partinfo>, every part behind medium strong constitutive promoter.

Bisphenol A:

  • BPA analysis with extraction and HPLC with UV detector leads to the same results as the analysis with LC-MS (except for low BPA concentrations -> LOD / LOQ of LC-MS is higher than of "normal" HPLC, compare figure to the right)
  • measuring of more samples from cultivations with BPA degrading BioBricks for further characterization
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