Team:Bielefeld-Germany/Labjournal

From 2011.igem.org

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

Contents

Week 1: 2nd - 8th May

Bisphenol A:

  • cloning of BBa_K123000 and BBa_K123001 behind weak (BBa_J23103) and medium strong (BBa_J23110) constitutive promoter (each part and both parts polycistronic)
  • cloning of fusion protein between BBa_K123000 and BBa_K123001, 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 Corynebacterium glutamicum and Corynebacterium crenatum
  • successful cloning of the S-layer gene of Brevibacterium 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 BBa_K157011 behind existing BPA degrading parts (for purification and testing of BBa_K123000 and BBa_K123001 in a cell free system)
  • HPLC results: Fusion protein between BBa_K123000 and BBa_K123001 can degrade BPA and seems to work better than the polycistronic version (compare Figure 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 C. glutamicum, 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)

Week 4: 23rd - 29th May

Bisphenol A:

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

Organizational:

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

Week 5: 30th May - 5th June

Bisphenol A:

Organizational:

Week 6: 6th - 12th June

Organizational:

Week 7: 13th - 19th June

S-layer:

  • successful fusion of modified cspB genes of C. glutamicum and C. halotolerans with a monomeric RFP (BBa_E1010) using Gibson assembly.
  • C.glutamicum:
  • C. halotolerans:
  • First expression of K525133 in E. coli KRX to test different induction time points und L-rhamnose concentrations. A higher inducer concentration results in a decreasing maximum and final optical density (OD600) but in a higher fluorescence level. The variations of the induction time point has no significant influence on growth and the progression of fluorescence.
Figure 2: Growth curve of E. coli KRX expressing K525133 using different inducer (L-rhamnose) concentrations and varying the time point of induction. Cultivations were carried out in LB medium. Inducing time points and inducer concentration are named with following indices: (E1) induced early (cultivation time = 2,75 h) L-rhamnose end concentration = 0,1 %; (E2) induced early (cultivation time = 2,75 h) L-rhamnose end concentration = 0,2 %; (L1) induced late (cultivation time = 4,25 h) L-rhamnose end concentration = 0,1 %; (L2) induced late (cultivation time = 4,25 h) L-rhamnose end concentration = 0,2 %
Figure 3: Fluorescence development of E. coli KRX expressing K525133 during cultivation using different inducer (L-rhamnose) concentrations and varying the time point of induction. Cultivations were carried out in LB medium. Inducing time points and inducer concentration are named with following indices: (E1) induced early (cultivation time = 2,75 h) L-rhamnose end concentration = 0,1 %; (E2) induced early (cultivation time = 2,75 h) L-rhamnose end concentration = 0,2 %; (L1) induced late (cultivation time = 4,25 h) L-rhamnose end concentration = 0,1 %; (L2) induced late (cultivation time = 4,25 h) L-rhamnose end concentration = 0,2 %

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

EXAMS !

Week 10: 4th July - 10th July

Bisphenol A:

  • experiments on the influence of temperature, promoter strength and the characteristics of the fusion protein BBa_K123000 || BBa_K123001 on BPA degradation

S-layer:

  • Expression of K525131 (with TAT-sequence and lipid anchor), K5252133 (with lipid anchor), K525232 (nothing); K525233 (with lipid anchor) and K525234 (with TAT-sequence) in E. coli KRX to test the functional efficiency of Corynebacterium TAT-sequence in E.coli and the effect of the autoinduction protocol. After one day of cultivation, an increased fluorescence in all periplasm fractions of E. coli expressing S-layer constructs with TAT-sequence could be recognized. This is an indication that the Corynebacterium TAT-signal sequence is functional in E. coli. Comparing the manual and the autoinduction protocol, no significant changes in fluorescence (after 14 h) are identifiable.
Figure 4: Relative fluorescence of the S-layer/mRFP fusion proteins in the cell suspension less the distinct fluorescence of E. coli KRX. All cultivations were carried out in LB medium at 37 ˚C except K525131Glc which was in autoinduction medium. The expression of the S-layers in LB medium were induced adding 0,1 % L-rhamnose at OD600 = 0,6.
Figure 5: Relative fluorescence of the S-layer/mRFP fusion proteins in the periplasm less the distinct fluorescence of E. coli KRX periplasm. All cultivations were carried out in LB medium at 37 ˚C except K525131Glc which was in autoinduction medium. The expression of the S-layers in LB medium were induced adding 0,1 % L-rhamnose at OD600 = 0,6.

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 fluorescent proteins and cleavage sites) from iGEM HQ arrived

S-layer:

  • our synthesized S-layers SgsE and SbpA finally arrived

Organizational:

Week 12: 18th July - 24th July

Organizational:

  • presenting our projects from 2010 and 2011 at the CeBiTec Symposium 2011 in Bielefeld
  • meeting with the iGEM teams from Delft/NL, Edinburgh/UK, Odense/DK. Freiburg/DE and Ljubljana/SL at the Symposium (Photos) was fun!

Bisphenol A / S-Layer:

  • removing illegal restriction sites to get valid BioBricks

NAD+ detection:

  • cloning the NAD+-dependent DNA 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
  • successful cloning of K525231 using Gibson assembly.
  • testing the basal transcription of E. coli KRX by adding inducer (L-rhamnose) or not. The increasing RFU per OD600 reveals the basal transcription of E. coli KRX. L-rhamnose in the medium leads to a ten times higher RFU per OD600.
Figure 6: Growth curves of E. coli KRX expressing fluorescence tagged S-layer constructs of C. glutamicum. Cultivations were carried out in autoinduction medium at 37 ˚C with and without adding inducer (L-rhamnose). The induced cultivations are marked with index I and the uninduced are marked with index U.
Figure 7: Growth curves of E. coli KRX expressing fluorescence tagged S-layer constructs of C. halotolerans. Cultivations were carried out in autoinduction medium at 37 ˚C with and without adding inducer (L-rhamnose). The induced cultivations are marked with index I and the uninduced are marked with index U.


Figure 8: Development of relative fluorescence per OD600 during cultivation of E. coli KRX expressing fluorescence tagged S-layer constructs of C.glutamicum and C. halotolerans. Cultivations were carried out in autoinduction medium at 37 ˚C.
Figure 9: Development of relative fluorescence per OD600 during uninduced cultivation of E. coli KRX containing fluorescence tagged S-layer gens constructs of C.glutamicum and C. halotolerans. Cultivations were carried out in autoinduction medium at 37 ˚C without adding inducer.
Figure 10: Development of relative fluorescence per OD600 in periplasm of E. coli KRX expressing fluorescence tagged S-layer constructs of C. glutamicum and C. halotolerans during cultivation. Cultivations were carried out in autoinduction medium at 37 ˚C.

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 (BBa_K123000 to BBa_K123001) can completely degrade BPA
  • Measuring characterization results for different BPA degrading BioBricks

NAD+ detection:

  • Successful characterization of two differently labeled (6-FAM or TAMRA with Dabcyl) molecular beacons as a preparation for the NAD+ bioassay including autonomously produced NAD+-dependent DNA ligase (BBa_K525710) from E. coli (results shown below)
Figure 11: 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 12: Emission spectra of 6-FAM labeled molecular beacon in its closed state (split target added) at an extinction wavelength 495 nm (n=3).


Figure 13: 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 14: Extinction spectra of 6-FAM labeled molecular beacon in its closed state (split target added) at an emission wavelength 530 nm (n=3).


Figure 15: Emission spectra of TAMRA labeled molecular beacon in its closed or open state (target added) at an extinction wavelength 544 nm (n=3).
Figure 16: Emission spectra of TAMRA labeled molecular beacon in its closed state (split target added) at an extinction wavelength 544 nm (n=3).


Figure 17: Extinction spectra of TAMRA labeled molecular beacon in its closed or open state (target added) at an emission wavelength 590 nm (n=3).
Figure 18: Extinction spectra of TAMRA labeled molecular beacon in its closed state (split target added) at an emission wavelength 590 nm (n=3).


Figure 19: 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 20: 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 21: 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 22: 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).


Figure 23: Thermal profile of 6-FAM labeled molecular beacon alone and with either target or split target added (n=5).
Figure 24: Imaging 6-FAM labeled molecular beacon alone and with either target or split target added.

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 25)
  • 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 model for BPA degradation by E. coli (compare Figure 26)
Figure 25: 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 (BBa_K123000), 512: polycistronic BBa_K123001 and BBa_K123000, 517: fusionprotein between BBa_K123001 and BBa_K123000, every part behind medium strong constitutive promoter.
Figure 26: 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 BBa_J23110. 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 KRX.

Week 16: 15th August - 21st August

S-layer:

  • finally found our S-layer proteins in E. coli by analyzing the range of sizes in the polyacrylamid with a MALDI-TOF method-> 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
    • some "metabolites" could also be found due to fragmentation during ionization of the product of BPA degradation
  • testing whether E. coli can grow on BPA as the only carbon source (on M9 plates)
    • seems like they can't

Week 17: 22nd August - 28th August

S-layer:

  • cultivation and test purification of SgsE fusion proteins (BBa_K525304, BBa_K525305 and BBa_K525306)
    • SDS-PAGEs showed inclusion bodies formation in E coli KRX expressing sgsE.

Bisphenol A:

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

Week 18: 29th August - 4th September

S-layer:

  • developing IEX clean-up for S-layers from Corynebacterium halotolerans
  • Searching new purification methods for SgsE fusion proteins.

Bisphenol A:

  • successful cloning of polycistronic FNR + BisdA + BisdB

NAD+ detection:

  • Successful overexpression of NAD+-dependent DNA ligase (BBa_K525710) in E. coli KRX and purification with Ni-NTA columns utilizing the protein`s C-terminal His-tag. Trying out utility for the NAD+ bioassay now.

Organizational:

Week 19: 5th September - 11th September

Fig. 27: Immobilization behaviour of fluorescent S-layer fusion protein SgsE :: mCitrine as a function of silica bead concentration. Data fitted with Michaelis-Menten function.

S-layer:

  • coating silica beads with fluorescent S-layer fusion protein SgsE | mCitrine, remove these proteins again and find them in SDS-PAGE
  • characterizing immobilization behaviour of S-layers on silica beads -> seems to work (Figure 27)
  • cultivate bigger amounts of and develop new purification strategy for S-layer fusion proteins
  • testing purification of S-layer fusion proteins with his-tag
  • cultivations of fusion protein between NADP+-ferredoxin oxidoreductase and SgsE / SbpA
  • also SbpA | mCitrine fusion protein

NAD+ detection:

  • Suitability of autonomously produced DNA ligase (BBa_K525710) from E. coli for the NAD+ bioassay could be demonstrated after treatment with NMN for deadenylation. Checking out the limit of detection right now.


Week 20: 12th September - 18th September

S-layer:

Bisphenol A:

  • successful cloning of fusion protein FNR + BisdA | BisdB and polycistronic FNR + BisdA + BisdB behind constitutive promoter -> characterizing these last constructs

NAD+ detection:

  • NAD+ assay works :)

Week 21: 19th September - 25th September

Organizational:

  • lab work is finished, the last measurements and cloning experiments into pSB1C3 are done
  • now it is time to fill the Wiki and Partsregistry with brilliant data until European Wikifreeze @ September 21st 11:59 pm EDT!

Week 22: 26th September - 2nd October

Preparation of the presentation and the poster for the European Regional Jamboree.

  • practicing the presentation with the Coryne and fermentation engineering working group as audience.

We are qualified for the final in Boston!!! Now labwork can continue.

Week 23: 3rd October - 9th October

Figure 28: LigA shows high selectivity for NAD+. The final concentration of all analytes was 100 nM. The responses were evaluated on the basis of the average fluorescence enhancement rate in a range of 200 s after addition of each analyte into the NAD+ bioassay. The dotted line marks the threshold indicating the intensity of background signal. All data are normalized to the NAD+ value.

S-layer:

NAD+ detection:

  • testing selectivity of LigA (BBa_K525710) for its substrate NAD+ to verify further applications of the molecular beacon based NAD+ bioassay dealing with NAD+ detection in analyte mixtures such as cell lysates.


Week 24: 10th October - 16th October

Figure 29: Fluorescence enhancement rate after addition of LDH reaction mix with various pyruvate concentrations into a LigA-dependent NAD+ bioassay.

S-layer:

  • first expression of K525422 in E. coli KRX and His-tag affinity chromatography based purification using denaturing conditions.
    • Test purifications were carried out using binding buffer with and without imidazole.
  • new inclusion body purification of K525305 and K525405 to get new biological material to test the further purification methods.
  • establishment of ion exchange chromatography (IEX) and hydrophobic interaction chromatography (HIC) as futher purification steps for S-layer proteins from Lysinibacillus sphaericus and Geobacillus stearothermophilus.
    • diethylaminoethyl cellulose (DEAE) as IEX and butyl sepharose™ 4 Fast Flow as HIC purification media.
  • test and scale-up expression of mCitrine|SbpA|His-tag (K525322) in E. coli KRX.
    • after enzymatic cell lysis with lysozyme and inclusion body purification highest luminescence was measured in the lysis supernatant. This implicates that the fusion protein does not form inclusion bodies.
  • successful cloning of fusion protein of CspB and luciferase with Gibson assembly

NAD+ detection:

  • the NAD+ bioassay was successfully coupled to the NADH-dependent reaction of lactic acid dehydrogenase and could therefore be used to quantify pyruvate

BPA-degradation:

Week 25: 17th October - 23th October

S-layer:

  • new expression of BBa_K525311 to develop an individual purification strategy without inclusion body purification.
  • testing a new purification strategy for the fusion protein of the SgsE and the firefly-luciferase based on the IEX and HIC
  • first expression of mCitrine|SbpA|His-tag (K525322) and successful His-tag affinity chromatography based purification using denaturing conditions.

NAD+ detection:

  • testing LigA for molecular cloning

Week 26: 24th October - 30th October

S-layer:

  • test expression of reductase|His-tag (K525534) in E. coli KRX. and testing non denaturating His-tag affinity chromatography.
  • recrystallization of mCitrine|SgsE (K525305) and mCitrine|SbpA (K525405) on silicon wafer using two different immobilization strategies.
  • trying to get pictures of the S-layer nanostructures on coated silicon wafers using reflection electron microscope (REM) and atomic force microscopy (AFM)
    • not enough time left to find a successful method