MARSS M odulated A cetosyringone R eceptor S ensor S ystem Aretz J., Brombacher E., Hänisch F., Kessler N, Lübke N.C., Marschall J., Neshat A., Tschapalda K., Unthan S., Walter F., Wolf T., Dr. Rückert C., Dr. Kalinowski J., Prof. Dr. Niehaus K. Freitag, 5. November 2010
Introduction Approach Results Publicity Conclusion Outlook Google Maps™-Kartenservice 2 Freitag, 5. November 2010
Approach Introduction Results Publicity Conclusion Outlook ‹ MARSS - Project idea 3 Freitag, 5. November 2010
Approach Introduction Results Publicity Conclusion Outlook ‹ MARSS - Project idea Biosensor based on a two- component regulatory sytem Graphic by Bianca Meyer, Westfalen Blatt August 4 th 2010 3 Freitag, 5. November 2010
Approach Introduction Results Publicity Conclusion Outlook ‹ MARSS - Project idea Directed evolution driven receptor modification to detect new substances 3 Freitag, 5. November 2010
Approach Introduction Results Publicity Conclusion Outlook ‹ Background Agrobacterium tumefaciens: a phyto pathogen A. Griffiths et al ., 2000 4 Freitag, 5. November 2010
Approach Introduction Results Publicity Conclusion Outlook ‹ Background Agrobacterium tumefaciens: a phyto pathogen A. Griffiths et al ., 2000 4 Freitag, 5. November 2010
Approach Introduction Results Publicity Conclusion Outlook ‹ Requirements for VirA activator 5 Freitag, 5. November 2010
Approach Introduction Results Publicity Conclusion Outlook ‹ Requirements for VirA activator 1. Phenol Acetosyringone 1-(4-hydroxy-3,5-dimethoxyphenyl)ethanone 5 Freitag, 5. November 2010
Approach Introduction Results Publicity Conclusion Outlook ‹ Requirements for VirA activator 1. Phenol 2. � Methoxy groups in ortho position to the hydroxyl group Acetosyringone 1-(4-hydroxy-3,5-dimethoxyphenyl)ethanone 5 Freitag, 5. November 2010
Approach Introduction Results Publicity Conclusion Outlook ‹ Requirements for VirA activator 1. Phenol 2. � Methoxy groups in ortho position to the hydroxyl group 3. � Acetyl- or Alkyl group in para position Acetosyringone 1-(4-hydroxy-3,5-dimethoxyphenyl)ethanone 5 Freitag, 5. November 2010
Approach Introduction Results Publicity Conclusion Outlook ‹ Potential compounds Acetosyringone Metanephrine Homovanillic acid Capsaicin Vanilmandelic acid Dopamine NCBI.gov 6 Freitag, 5. November 2010
Approach Introduction Results Publicity Conclusion Outlook ‹ Potential compounds Capsaicin NCBI.gov 6 Freitag, 5. November 2010
Results Introduction Approach Publicity Conclusion Outlook Transfering modified VirA/G signaling system to E. coli 7 Freitag, 5. November 2010
Results Introduction Approach Publicity Conclusion Outlook Transfering modified VirA/G signaling system to E. coli 7 Freitag, 5. November 2010
Results Introduction Approach Publicity Conclusion Outlook Transfering modified VirA/G signaling system to E. coli 7 Freitag, 5. November 2010
Results Introduction Approach Publicity Conclusion Outlook Transfering modified VirA/G signaling system to E. coli 7 Freitag, 5. November 2010
Results Introduction Approach Publicity Conclusion Outlook Transfering modified VirA/G signaling system to E. coli 7 Freitag, 5. November 2010
Results Introduction Approach Publicity Conclusion Outlook Transfering modified VirA/G signaling system to E. coli 8 Freitag, 5. November 2010
Results Introduction Approach Publicity Conclusion Outlook Characterization of final mRFP system 1200 1000 800 -1 [-] RFU OD 600 600 400 200 0 0 2 4 6 8 10 12 9 Freitag, 5. November 2010
Results Introduction Approach Publicity Conclusion Outlook Characterization of final mRFP system 1200 1000 800 -1 [-] RFU OD 600 600 400 Basal expression occurs 200 0 0 2 4 6 8 10 12 9 Freitag, 5. November 2010
Results Introduction Approach Publicity Conclusion Outlook Characterization of final mRFP system 1200 1000 Induction is evident 800 -1 [-] RFU OD 600 600 400 Basal expression occurs 200 0 0 2 4 6 8 10 12 9 Freitag, 5. November 2010
Results Introduction Approach Publicity Conclusion Outlook Characterization of final sensing system Induction with acetosyringone q P,n: specific production rate normalized to uninduced status q p c: specific production rate for induced production state q p o: specific production rate for uninduced production state At least three biological replicates mRFP 3,5 Switch point: 26.5 µM Top asymptote: 2.62 3,0 2,5 -1 ] q P,n [h 2,0 1,5 1,0 0,5 0 1 2 3 10 Freitag, 5. November 2010
Results Introduction Approach Publicity Conclusion Outlook Characterization of final sensing system Induction with acetosyringone Φ I : ratio of product per cell induced to uninduced i: induced u: uninduced At least three biological replicates mRFP 2,5 Switch point: 26.5 µM Top asymptote: 2.62 2,0 I [-] Luc 1,5 Φ Switch point: 31.6 µM Top asymptote: 2.16 1,0 0 1 2 3 10 Freitag, 5. November 2010
Results Introduction Approach Publicity Conclusion Outlook Sensitivity Tuner amplified luc signal 11 Freitag, 5. November 2010
Results Introduction Approach Publicity Conclusion Outlook Sensitivity Tuner amplified luc signal 11 Freitag, 5. November 2010
Results Introduction Approach Publicity Conclusion Outlook Sensitivity Tuner amplified luc signal 1 1I III 11 Freitag, 5. November 2010
Results Introduction Approach Publicity Conclusion Outlook Directed Evolution Screening plasmid Plasmid for mutation 12 Freitag, 5. November 2010
Results Introduction Approach Publicity Conclusion Outlook Directed Evolution 100 80 60 S [%] 40 Φ 20 0 0 1 2 10 10 10 13 Freitag, 5. November 2010
Results Introduction Approach Publicity Conclusion Outlook Directed Evolution 1. Library construction virA error prone PCR x x restriction ligation virA* -library x x ColE1-ori 14 Freitag, 5. November 2010
Results Introduction Approach Publicity Conclusion Outlook Directed Evolution 1. Library construction 2. Primary selection x x virA* -Library x x Electroporation PvirB KanR Selection: Kan, Amp, Cm & Dopamin R6K-ori VirA substrate candidates Capsaicin E. coli EC100D Homovanillic acid 3-Methoxytyramine x x 15 Freitag, 5. November 2010
Results Introduction Approach Publicity Conclusion Outlook Directed Evolution 1. Library construction 3. Isolation of mutated receptor 2. Primary selection Miniprep Electroporation in TOP10 pir- TOP10 pir- EC100D pir+ Ampicillin TOP10 pir- 16 Freitag, 5. November 2010
Publicity Introduction Approach Results Conclusion Outlook Biosensor for spiciness Capsaicin Image by Florian Schmale 17 Freitag, 5. November 2010
Publicity Introduction Approach Results Conclusion Outlook Image by Alexandra Kardinar and Volker Schlecht (DIE ZEIT, May 27 th 2010) 18 Freitag, 5. November 2010
Publicity Introduction Approach Results Conclusion Outlook 19 Freitag, 5. November 2010
Publicity Introduction Approach Results Conclusion Outlook * Printing press Leading article Advanced notice * Online * Television Article initiated Documentary „DNA cooking“ [tba 2011] * Radio Feature Nano Interview at biotechnologie.tv 20 Freitag, 5. November 2010
Publicity Introduction Approach Results Conclusion Outlook Public Discussion Science Café Only fruit juice- no bacteria 21 Freitag, 5. November 2010
Conclusion Introduction Approach Results Publicity Outlook 7. Conclusion Safety Use existing sensor system BioBricks in E. coli Build up a read out system - proportional to ligand concentration Adaptation of existing sensitivity tuner Build up a screening system to select positive receptor changes Develop a modification system 8 new BioBricks + 16 composite (sequenced, characterized and working) Defining spiciness since 2010 22 Freitag, 5. November 2010
Conclusion Introduction Approach Results Publicity Outlook 7. Conclusion Safety Use existing sensor system BioBricks in E. coli Build up a read out system - proportional to ligand concentration Adaptation of existing sensitivity tuner Build up a screening system to select positive receptor changes Develop a modification system 8 new BioBricks + 16 composite (sequenced, characterized and working) Defining spiciness since 2010 22 Freitag, 5. November 2010
Conclusion Introduction Approach Results Publicity Outlook 7. Conclusion Safety Use existing sensor system BioBricks in E. coli Build up a read out system - proportional to ligand concentration Adaptation of existing sensitivity tuner Build up a screening system to select positive receptor changes Develop a modification system 8 new BioBricks + 16 composite (sequenced, characterized and working) Defining spiciness since 2010 22 Freitag, 5. November 2010
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