MARSS M odulated A cetosyringone R eceptor S ensor S ystem Aretz J., - - PowerPoint PPT Presentation
MARSS M odulated A cetosyringone R eceptor S ensor S ystem Aretz J., Brombacher E., Hnisch F., Kessler N, Lbke N.C., Marschall J., Neshat A., Tschapalda K., Unthan S., Walter F., Wolf T., Dr. Rckert C., Dr. Kalinowski J., Prof. Dr. Niehaus
MARSS Modulated Acetosyringone Receptor Sensor System
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
Approach Results Publicity Conclusion Outlook Introduction
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Google Maps™-Kartenservice
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Biosensor based on a two- component regulatory sytem
Graphic by Bianca Meyer, Westfalen Blatt August 4th 2010
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Directed evolution driven receptor modification to detect new substances
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Agrobacterium tumefaciens: a phyto pathogen
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Agrobacterium tumefaciens: a phyto pathogen
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Acetosyringone 1-(4-hydroxy-3,5-dimethoxyphenyl)ethanone
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Acetosyringone 1-(4-hydroxy-3,5-dimethoxyphenyl)ethanone
2.Methoxy groups in ortho position to the hydroxyl group
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Introduction Results Publicity Conclusion Outlook Approach
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Acetosyringone 1-(4-hydroxy-3,5-dimethoxyphenyl)ethanone
2.Methoxy groups in ortho position to the hydroxyl group 3.Acetyl- or Alkyl group in para position
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Introduction Results Publicity Conclusion Outlook Approach
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NCBI.gov
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NCBI.gov
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Introduction Approach Publicity Conclusion Outlook Results
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Transfering modified VirA/G signaling system to E. coli
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Introduction Approach Publicity Conclusion Outlook Results
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Transfering modified VirA/G signaling system to E. coli
Freitag, 5. November 2010
Introduction Approach Publicity Conclusion Outlook Results
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Transfering modified VirA/G signaling system to E. coli
Freitag, 5. November 2010
Introduction Approach Publicity Conclusion Outlook Results
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Transfering modified VirA/G signaling system to E. coli
Freitag, 5. November 2010
Introduction Approach Publicity Conclusion Outlook Results
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Transfering modified VirA/G signaling system to E. coli
Freitag, 5. November 2010
Introduction Approach Publicity Conclusion Outlook Results
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Transfering modified VirA/G signaling system to E. coli
Freitag, 5. November 2010
Introduction Approach Publicity Conclusion Outlook Results
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2 4 6 8 10 12 200 400 600 800 1000 1200
RFU OD600
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Introduction Approach Publicity Conclusion Outlook Results
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Basal expression occurs
2 4 6 8 10 12 200 400 600 800 1000 1200
RFU OD600
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Introduction Approach Publicity Conclusion Outlook Results
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Basal expression occurs Induction is evident
2 4 6 8 10 12 200 400 600 800 1000 1200
RFU OD600
Freitag, 5. November 2010
Introduction Approach Publicity Conclusion Outlook Results
1 2 3 0,5 1,0 1,5 2,0 2,5 3,0 3,5
qP,n [h
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Switch point: 26.5 µM Top asymptote: 2.62
Induction with acetosyringone qP,n: specific production rate normalized to uninduced status qp c: specific production rate for induced production state qp o: specific production rate for uninduced production state
mRFP
At least three biological replicates
Freitag, 5. November 2010
Introduction Approach Publicity Conclusion Outlook Results
1 2 3 1,0 1,5 2,0 2,5
I [-]
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Switch point: 26.5 µM Top asymptote: 2.62 Switch point: 31.6 µM Top asymptote: 2.16
mRFP Luc
Induction with acetosyringone ΦI: ratio of product per cell induced to uninduced i: induced u: uninduced
At least three biological replicates
Φ
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Introduction Approach Publicity Conclusion Outlook Results
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Freitag, 5. November 2010
Introduction Approach Publicity Conclusion Outlook Results
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Introduction Approach Publicity Conclusion Outlook Results
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1 1I III
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Introduction Approach Publicity Conclusion Outlook Results
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Screening plasmid Plasmid for mutation
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Introduction Approach Publicity Conclusion Outlook Results
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10 10
110
220 40 60 80 100
S [%]
Φ
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Introduction Approach Publicity Conclusion Outlook Results
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x x x x virA error prone PCR restriction ligation virA*-library ColE1-ori
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Introduction Approach Publicity Conclusion Outlook Results
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Dopamin Capsaicin Homovanillic acid 3-Methoxytyramine
x x virA*-Library KanR Electroporation
x xPvirB R6K-ori Selection: Kan, Amp, Cm & VirA substrate candidates
x xFreitag, 5. November 2010
Introduction Approach Publicity Conclusion Outlook Results
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EC100D pir+ Miniprep TOP10 pir- Electroporation in TOP10 pir- Ampicillin TOP10 pir-
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Introduction Results Approach Conclusion Outlook Publicity
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Image by Florian Schmale
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Introduction Results Approach Conclusion Outlook Publicity
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Image by Alexandra Kardinar and Volker Schlecht (DIE ZEIT, May 27th 2010)
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Introduction Results Approach Conclusion Outlook Publicity
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Introduction Results Approach Conclusion Outlook Publicity
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* Printing press
Leading article Advanced notice
* Online
Article initiated
* Radio * Television
Documentary „DNA cooking“ [tba 2011] Feature Nano Interview at biotechnologie.tv
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Introduction Results Approach Conclusion Outlook Publicity
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Only fruit juice- no bacteria
Freitag, 5. November 2010
Introduction Results Publicity Approach Outlook 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)
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Defining spiciness since 2010
Freitag, 5. November 2010
Introduction Results Publicity Approach Outlook 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)
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Defining spiciness since 2010
Freitag, 5. November 2010
Introduction Results Publicity Approach Outlook 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)
22
Defining spiciness since 2010
Freitag, 5. November 2010
Introduction Results Publicity Approach Outlook 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)
22
Defining spiciness since 2010
Freitag, 5. November 2010
Introduction Results Publicity Approach Outlook 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)
22
Defining spiciness since 2010
Freitag, 5. November 2010
Introduction Results Publicity Approach Outlook 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)
22
Defining spiciness since 2010
Freitag, 5. November 2010
Introduction Results Publicity Approach Outlook 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)
22
Defining spiciness since 2010
Freitag, 5. November 2010
Introduction Results Publicity Approach Outlook 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)
22
Defining spiciness since 2010
Freitag, 5. November 2010
Introduction Results Publicity Approach Outlook 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)
22
Defining spiciness since 2010
Freitag, 5. November 2010
Introduction Results Publicity Approach Outlook 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)
22
Defining spiciness since 2011!
Freitag, 5. November 2010
Introduction Results Publicity Conclusion Approach Outlook
Continue error prone PCR to validate the proof of concept Minimize measuring time Reduce basal expression of virB promoter driven gene Apply concept to other receptors or adapt VirA to compounds of more economic or medical relevant substances
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Freitag, 5. November 2010
Bioinformatics of Signaling Networks Christina Sophie Lübke Xi Chen Varun Shah Marco La Russa Teresa Quiroga Puertas Dimitri Miller Jana Blotenberg iGEM Team ZJU-China 24
2010.igem.org/Team:Bielefeld-Germany
Freitag, 5. November 2010