Team University of Göttingen
Team University of Göttingen In brightest day, in darkest night, No pathogen shall escape my sight. New antibiotics I will find, My enemies forever I bind!
Team University of Göttingen
Team University of Göttingen
The beast and its Achilles’ heel A novel target to fight multi-resistant pathogenic bacteria
The beast and its Achilles’ heel A novel target to fight multi-resistant pathogenic bacteria
Green Coli´s appartment Laboratory
The threat The US Threat Report : Yearly 2 million persons infected by resistant bacteria 23,000 deaths yearly
Staphylococcus aureus Bacteria on 25-30% of all humans Harmless for healthy individuals Can cause illness in weak or immune- suppressed individuals Is relatively easy to treat with antibiotics
Staphylococcus aureus Bacteria on 25-30% of all humans Harmless for healthy individuals Can cause illness in weak or immune- suppressed individuals Is relatively easy to treat with antibiotics But…
Antibiotic resistances S. aureus is a paradigm for bacteria that acquire antibiotic resistances Some strains are even untreatable
Antibiotic resistances S. aureus is a paradigm for bacteria that acquire antibiotic resistances Some strains are even untreatable So why not just develop new antibiotics? Known targets have been exhausted
Antibiotic resistances So why not just develop new antibiotics? Known targets have been exhausted
Green Coli´s appartment Laboratory
Human practice
Green Coli begins
Green Coli begins
Our aim Potential Substances Substance works! Not working! Antibiotic Detector
Green Coli´s appartment Laboratory
Our lab
Targets for antibiotics have to fulfill certain criteria Essential No homolog in humans Pleiotropic Conserved in pathogens Dr. Sven Halbedel, Robert-Koch-Institute Wernigerode
Cyclic di-AMP, a novel essential signaling nucleotide Staphylococcus Streptococcus aureus pneumoniae DAC c-di-AMP Mycobacterium Listeria tuberculosis monocytogenes
Our project: Tackling c-di-AMP as a target for antibiotics Construction of an antibiotic screening system in E. coli - based on known c-di- AMP sensors Identification of new c-di-AMP sensors for an alternative screening system Characterization of diadenylate cyclases for in vitro and in silico screening
Our project: Tackling c-di-AMP as a target for antibiotics Construction of an antibiotic screening system in E. coli - based on known c-di- AMP sensors Identification of new c-di-AMP sensors for an alternative screening system Characterization of diadenylate cyclases for in vitro and in silico screening Reporter Team!
DarR, a transcription factor sensing c-di-AMP Identified in Mycobacterium Transcriptional repressor Binds to DarR operator c-di-AMP stimulates binding DarR binding sequence N C DNA-binding c-di-AMP ATACTACGGAGTAT
A c-di-AMP reporter system based on DarR
A c-di-AMP reporter system based on DarR Output Output Biobricks we used: BBa_J23117; BBa_J23110; BBa_B0034; BBa_B0015; BBa_E0240
A c-di-AMP reporter system based on DarR Output Output Our new biobricks: BBa_K1045000 DarR operator BBa_K1045001 DarR repressor
A long, long way to the DarR reporter system Weak promoter Strong promoter Operator DarR reporter gfp darR system in pSB1C3 BBa_K1045017
A long, long way to the DarR reporter system Weak promoter Strong promoter operator DarR reporter darR gfp system in pSB1C3
Testing the DarR reporter system E. coli + BBa_K1045013 E. coli + BBa_K1045017 DarR is expressed and active as a repressor in E. coli
It’s worth to improve the reporter system! “I‘m total tally ly happy py that at the the iGEM Team m Gött ttingen ngen deci cided ded to to develop elop an in vivo vo scr creen ening ing system tem !“ Prof. Dr. Heike Brötz-Österhelt, HHU Düsseldorf
Our project: Tackling c-di-AMP as a target for antibiotics Construction of an antibiotic screening system in E. coli - based on known c-di- AMP sensors Identification of new c-di-AMP sensors for an alternative screening system Characterization of diadenylate cyclases Array Team! for in vitro and in silico screening
Identification of c-di-AMP-responsive genes in B. subtilis Cooperation with iGEM Team Groningen (Netherlands) Microarray: RNA from different Bacillus subtilis strains wild type c-di-AMP overproducer
A c-di-AMP-binding riboswitch regulates ydaO transcription Promoter Riboswitch ydaO ORF ON OFF ydaO riboswitch
Expression analysis of ydaO low c-di-AMP/high c-di-AMP 6 5 4 Fold change 3 2 1 0 ydaO control ydaO expression was up-regulated at low c-di-AMP levels
A riboswitch reporter system
A riboswitch reporter system Output Output Biobricks we used: BBa_E0020
A riboswitch reporter system Output Output ydaO riboswitch with Our new BBa_K1045004; BBa_K1045005; different flanking biobricks: BBa_K1045006; BBa_K1045007 regions
Testing the riboswitch reporter system E. coli transformed with BBa_K1045002 - c-di-AMP + c-di-AMP The ydaO promoter from Bacillus works in E. coli
Our project: Tackling c-di-AMP as a target for antibiotics Construction of an antibiotic screening system in E. coli - based on known c-di- AMP sensors DAC Team! Identification of new c-di-AMP sensors for an alternative screening system Characterization of diadenylate cyclases for in vitro and in silico screening
Characterization of L. monocytogenes DAC DAC with N-terminal affinity-tag Our new biobricks: BBa_K1045003 Lmo DAC
Purification of DAC Co 10 mg/ml DAC was purified in high amounts!
Activity of DAC in vivo 0,15 [µmol/mg protein] 0,1 c-di-AMP DAC 0,05 ND 0 control + DAC DAC is highly active in vivo
Activity of DAC in vitro 5 μ M DAC; pH 8 20 c-di-AMP [ μ M] 10 DAC 0 0 100 200 300 incubation time [min] DAC is active in vitro
Crystallization of DAC DAC domain
Crystallization of the DAC domain DAC domain The first st crysta tals ls!
Crystallization of DAC DAC domain A 2 2.8 Å di diffr fraction action pat attern rn!! !! The first crystals!
Crystallization of DAC ? DAC domain The first crystals! A 2.8 Å diffraction pattern!!
Crystallization of DAC A crys ystal al struc ucture!! ture!! !
Three ways to find new antibiotics in silico DAC in vitro in vivo
An in vitro screening system for Prof. Dr. Benkovic …? “ With th this enzyme me, , - DarR I wan I ant to to sc screen my my subs su bstan ance ce libr brary ary !“ Prof. Dr. Stephen Benkovic, Pennsylvania State University
Our biobricks Registry # Name New Biobricks DarR Protein, Inversed BBa_K1045001 DarR operator BBa_K1045000 YdaO Riboswitch with regulative flanking regions BBa_K1045004 YdaO Riboswitch only with 3`flanking region BBa_K1045005 YdaO Riboswitch only with 5` flanking region BBa_K1045006 YdaO Riboswitch without flanking regions BBa_K1045007 Diadenylate cyclase domain of L. monocytogenes cdaA BBa_K1045003 Improved Biobricks BBa_J23117 cyclic di amp acterim reverse BBa_K1045011 BBa_B0034 reverse BBa_K1045010 BBa_B0015 reverse BBa_K1045009 Composite Parts composite part BBa_B0015 reverse-DarR reverse - BBa_B0034 reverse-BBa_J23117 reverse-BBa_J23110-DarR operator - Bba_E0240 (DarR reporter system, weak promoter) BBa_K1045017 CFP Reporter under control of the c-di-AMP-dependent YdaO Riboswitch BBa_K1045002 Intermediate Parts composite part BBa_J23110 with DarR operator BBa_K1045012 composite part BBa_B0034 reverse-BBa_J23117 reverse-BBa_J23110-DarR operator - BBa_E0240 (6.1) BBa_K1045015 composite part BBa_B0015 reverse-DarR reverse BBa_K1045016 composite part BBa_J23117 reverse-BBa_J23110-DarR operator - Bba_E0240 BBa_K1045014 composite part BBa_J23110-DarR operator - Bba_E0240 BBa_K1045013
Green Coli´s appartment Laboratory
Perspectives High-throughput screening-system Thousands of substances can be tested in one run Potential candidates can be further investigated Saves human lives through new antibiotics
Our sponsors
Acknowledgements Dr. Achim Dickmanns Dr. Piotr Neumann Annette Garbe Our Supervisors
The beast and its Achilles’ heel Thank you for your interest!
The beast and its Achilles’ heel Thank you for your interest! Keep The Green Light Glowing!
The beast and its Achilles’ heel Thank you for your interest! Keep The Green Light Glowing!
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