Team University of Gttingen Team University of Gttingen In - - PowerPoint PPT Presentation

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Team University of Gttingen Team University of Gttingen In - - PowerPoint PPT Presentation

Oct 31, 2023 46 likes •683 views

Team University of Gttingen Team University of Gttingen 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 Gttingen Team University of

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Team University of Göttingen

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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!

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Team University of Göttingen

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Team University of Göttingen

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The beast and its Achilles’ heel

A novel target to fight multi-resistant pathogenic bacteria

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The beast and its Achilles’ heel

A novel target to fight multi-resistant pathogenic bacteria

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Green Coli´s appartment

Laboratory

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The threat

The US Threat Report: Yearly 2 million persons infected by resistant bacteria 23,000 deaths yearly

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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

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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…

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Antibiotic resistances

  • S. aureus is a paradigm for bacteria that

acquire antibiotic resistances Some strains are even untreatable

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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

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Antibiotic resistances

So why not just develop new antibiotics? Known targets have been exhausted

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Green Coli´s appartment

Laboratory

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Human practice

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Green Coli begins

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Green Coli begins

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Our aim

Antibiotic Detector Potential Substances Substance works! Not working!

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Green Coli´s appartment

Laboratory

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Our lab

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Essential No homolog in humans Pleiotropic Conserved in pathogens

  • Dr. Sven Halbedel,

Robert-Koch-Institute Wernigerode

Targets for antibiotics have to fulfill certain criteria

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c-di-AMP

Streptococcus pneumoniae Listeria monocytogenes Staphylococcus aureus Mycobacterium tuberculosis

Cyclic di-AMP, a novel essential signaling nucleotide

DAC

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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

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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

Reporter Team!

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Identified in Mycobacterium Transcriptional repressor Binds to DarR operator c-di-AMP stimulates binding

DNA-binding c-di-AMP

N C

DarR binding sequence ATACTACGGAGTAT

DarR, a transcription factor sensing c-di-AMP

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A c-di-AMP reporter system based on DarR

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Output Output

A c-di-AMP reporter system based on DarR

Biobricks we used: BBa_J23117; BBa_J23110; BBa_B0034; BBa_B0015; BBa_E0240

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Our new biobricks: BBa_K1045000 DarR operator BBa_K1045001 DarR repressor Output

A c-di-AMP reporter system based on DarR

Output

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A long, long way to the DarR reporter system darR gfp

DarR reporter system in pSB1C3

Strong promoter Operator Weak promoter BBa_K1045017

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A long, long way to the DarR reporter system

darR gfp

DarR reporter system in pSB1C3

Strong promoter

  • perator

Weak promoter

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  • E. coli + BBa_K1045013
  • E. coli + BBa_K1045017

Testing the DarR reporter system

DarR is expressed and active as a repressor in E. coli

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“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

It’s worth to improve the reporter system!

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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

Array Team!

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Cooperation with iGEM Team Groningen (Netherlands)

Microarray: RNA from different Bacillus subtilis strains wild type c-di-AMP overproducer

Identification of c-di-AMP-responsive genes in B. subtilis

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ydaO riboswitch

A c-di-AMP-binding riboswitch regulates ydaO transcription

Promoter Riboswitch ydaO ORF

ON OFF

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Expression analysis of ydaO

ydaO expression was up-regulated at low c-di-AMP levels

1 2 3 4 5 6 Fold change

low c-di-AMP/high c-di-AMP ydaO control

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A riboswitch reporter system

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A riboswitch reporter system

Biobricks we used: BBa_E0020 Output Output

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Our new biobricks: BBa_K1045004; BBa_K1045005; BBa_K1045006; BBa_K1045007 ydaO riboswitch with different flanking regions

A riboswitch reporter system

Output Output

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The ydaO promoter from Bacillus works in E. coli

Testing the riboswitch reporter system

  • E. coli transformed with BBa_K1045002
  • c-di-AMP

+ c-di-AMP

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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

DAC Team!

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DAC with N-terminal affinity-tag

Characterization of

  • L. monocytogenes DAC

Our new biobricks: BBa_K1045003 Lmo DAC

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Purification of DAC

10 mg/ml

DAC was purified in high amounts!

Co

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Activity of DAC in vivo

0,05 0,1 0,15

control + DAC c-di-AMP

[µmol/mg protein] ND

DAC is highly active in vivo

DAC

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Activity of DAC in vitro

DAC is active in vitro

10 20 100 200 300

c-di-AMP [μM]

incubation time [min]

5 μM DAC; pH 8

DAC

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DAC domain

Crystallization of DAC

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DAC domain

Crystallization of the DAC domain

The first st crysta tals ls!

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A 2 2.8 Å di diffr fraction action pat attern rn!! !!

The first crystals!

DAC domain

Crystallization of DAC

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The first crystals! A 2.8 Å diffraction pattern!!

DAC domain

Crystallization of DAC

?

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A crys ystal al struc ucture!! ture!!! Crystallization of DAC

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Three ways to find new antibiotics

in silico in vitro in vivo DAC

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  • DarR

“With th this enzyme me, , I I wan ant to to sc screen my my su subs bstan ance ce libr brary ary!“

  • Prof. Dr. Stephen Benkovic,

Pennsylvania State University

An in vitro screening system for

  • Prof. Dr. Benkovic…?
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Registry # Name New Biobricks

BBa_K1045001

DarR Protein, Inversed

BBa_K1045000

DarR operator

BBa_K1045004

YdaO Riboswitch with regulative flanking regions

BBa_K1045005

YdaO Riboswitch only with 3`flanking region

BBa_K1045006

YdaO Riboswitch only with 5` flanking region

BBa_K1045007

YdaO Riboswitch without flanking regions

BBa_K1045003

Diadenylate cyclase domain of L. monocytogenes cdaA

Improved Biobricks

BBa_K1045011

BBa_J23117 cyclic di amp acterim reverse

BBa_K1045010

BBa_B0034 reverse

BBa_K1045009

BBa_B0015 reverse

Composite Parts

BBa_K1045017

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_K1045002

CFP Reporter under control of the c-di-AMP-dependent YdaO Riboswitch

Intermediate Parts

BBa_K1045012

composite part BBa_J23110 with DarR operator

BBa_K1045015

composite part BBa_B0034 reverse-BBa_J23117 reverse-BBa_J23110-DarR operator - BBa_E0240 (6.1)

BBa_K1045016

composite part BBa_B0015 reverse-DarR reverse

BBa_K1045014

composite part BBa_J23117 reverse-BBa_J23110-DarR operator - Bba_E0240

BBa_K1045013

composite part BBa_J23110-DarR operator - Bba_E0240

Our biobricks

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Green Coli´s appartment

Laboratory

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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

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Our sponsors

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Acknowledgements

Annette Garbe

  • Dr. Piotr Neumann
  • Dr. Achim Dickmanns

Our Supervisors

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The beast and its Achilles’ heel

Thank you for your interest!

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The beast and its Achilles’ heel

Thank you for your interest! Keep The Green Light Glowing!

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The beast and its Achilles’ heel

Thank you for your interest! Keep The Green Light Glowing!