Team:Bielefeld-Germany/Labjournal

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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 BPA detection
  • expression of the successfully assembled BPA degrading BioBricks in E. coli TOP10

S-layer:

  • successful PCR on the S-layer genes of Brevibacterium flavum and Corynebacterium crenatum
  • successful cloning of the S-layer gene of B. flavum without TAT-sequence

Organizational:

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:

Organizational:

  • moving to our own room in the CeBiTec


Week 3: 16th - 22nd may

Bisphenol A:

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:


Week 6: 6th - 12th june

Organizational:


Week 7: 13th - 19th june

S-Layer:

  • B. flavum:
    • K525131: fusion of cspB including lipid anchor and TAT-sequence (K525101) with BBa_E1010
    • K525133: fusion of cspB including lipid anchor without TAT-sequence (K525103) with BBa_E1010
  • C. halotolerans:
    • K525233: fusion of cspB including lipid anchor without TAT-sequence (K525203) with BBa_E1010
    • K525224: fusion of cspB including TAT-sequence without lipid anchor (K525204) with BBa_E1010


Bisphenol A:

  • cloning of NADP oxidoreductase in pJET1.2 finally successful -> waiting for sequencing results to remove illegal restriction sites

Week 8: 20th - 26th june

S-layer:


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:

Week 10: 4th july - 10th july

Bisphenol A:

  • experiments on the influence of temperature, promotor intensity and the characteristics of the fusion protein <partinfo>K123000</partinfo> || <partinfo>K123001</partinfo> on BPA degradation

Organizational:

  • presenting our posters from the teams of 2010 and 2011 at the congress 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:


Week 12: 18th july - 24th july

Organizational:

  • presenting our projects from 2010 and 2011 at the 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 (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
Figure 2: Emission spectra of 6-FAM labeled Molecular Beacon in its closed and open state (target added) at an extinction wavelength 495 nm (n=3).
Figure 3: Emission spectra of 6-FAM labeled Molecular Beacon in its closed state (split target added) at an extinction wavelength 495 nm (n=3).


Figure 4: Extinction spectra of 6-FAM labeled Molecular Beacon in its closed and open state (target added) at an emission wavelength 530 nm (n=3).
Figure 5: Extinction spectra of 6-FAM labeled Molecular Beacon in its closed state (split target added) at an emission wavelength 530 nm (n=3).


Figure 6: Emission spectra of TAMRA labeled Molecular Beacon in its closed or open state (target added) at an extinction wavelength 544 nm (n=3).
Figure 7: Emission spectra of TAMRA labeled Molecular Beacon in its closed state (split target added) at an extinction wavelength 544 nm (n=3).


Figure 8: Extinction spectra of TAMRA labeled Molecular Beacon in its closed or open state (target added) at an emission wavelength 590 nm (n=3).
Figure 9: Extinction spectra of TAMRA labeled Molecular Beacon in its closed state (split target added) at an emission wavelength 590 nm (n=3).


Figure 10: Signal-to-background ratio (S/B) determination of 6-FAM labeled Molecular Beacon in its closed or open state at an extinction wavelength 495 nm and emission wavelength 530 nm. Molecular Beacons and the target were added one after another (see gaps) each after equilibrium was reached. Calculated S/B: 45,52 (n=3).
Figure 11: Signal-to-background ratio (S/B) determination of 6-FAM labeled Molecular Beacon in its closed state at an extinction wavelength 495 nm and emission wavelength 530 nm. Molecular Beacons and the split target were added one after another (see gaps) each after equilibrium was reached. Calculated S/B: 3,36 (n=3).


Figure 12: Signal-to-background ratio (S/B) determination of TAMRA labeled Molecular Beacon in its closed or open state at an extinction wavelength 552 nm and emission wavelength 590 nm. Molecular Beacons and the target were added one after another (see gaps) each after equilibrium was reached. Calculated S/B: 18,21 (n=3).
Figure 13: Signal-to-background ratio (S/B) determination of TAMRA labeled Molecular Beacon in its closed state at an extinction wavelength 552 nm and emission wavelength 590 nm. Molecular Beacons and the split target were added one after another (see gaps) each after equilibrium was reached. Calculated S/B: 2,31 (n=3).


File:Bielefeld-Germany-2011
Figure 14: Thermal profile of 6-FAM labeled Molecular Beacon alone and with either target or split target added (n=5).


Week 15: 8th august - 14th august

Bisphenol A:

  • BPA analysis with extraction and HPLC with UV detector leads to very similar results as the analysis with LC-MS (except for low BPA concentrations -> LOD / LOQ of LC-MS is lower than that of "normal" HPLC, compare figure 15)
  • measuring of more samples from cultivations with BPA degrading BioBricks for further characterization
  • we discovered some interesting results in our MS data - soon more
  • testing methods to purify his-tagged BisdA and BisdB for cell free BPA degradation and further characterization of these proteins
  • testing the influence of BPA on the growth of E. coli
  • developing a modell for BPA degradation by E. coli (compare fig. 16)
Figure 15: 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.
Figure 16: Modelling of BPA degradation (filled squares) by and OD600 (open squares) of E. coli KRX carrying genes for BisdA and BisdB (polycistronic bisdAB (black) and fusion protein between BisdA and BisdB (red)) behind the medium strong promoter <partinfo>J23110</partinfo>. Cultivations were carried out at 30 °C in LB + Amp + BPA medium for 24 h with automatic sampling every three hours in 300 mL shaking flasks without baffles with silicon plugs. Three biological replicates were analysed.


S-layer:

  • MALDI-TOF analysis of SDS-PAGEs to characterize the function of the TAT-sequence and the lipid anchor of PS2 (encoded by cspB gene) in E. coli

Week 16: 15th august - 21st august

S-layer:

  • finally found our S-layer proteins in E. coli -> now we can plan purification strategy
  • fusion of FPs to sgsE successful

Bisphenol A:

  • all metabolites of natural BPA degradation pathway found during degradation of BPA in E. coli by LC-MS -> now MS/MS to check structure of these metabolites to be sure
  • testing whether E. coli can grow on BPA as the only carbon source (on M9 plates)


Week 17: 22nd august - 28th august

S-layer:

  • cultivation and purification of sgsE fusion proteins

Bisphenol A:

  • purification of his-tagged BisdA and BisdB
  • first characterization results entered into partsregistry
  • cloning of fusion protein FNR:BisdA:BisdB successful
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