Stalled antibiotics development A solved problem No new classes of - - PowerPoint PPT Presentation

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Stalled antibiotics development A solved problem No new classes of - - PowerPoint PPT Presentation

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Stalled antibiotics development A solved problem No new classes of antibiotics for 30 years Stalled antibiotics development A solved problem No new classes of antibiotics for 30 years Walsh & Fischbach (2009) Sci.

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Stalled antibiotics development

  • ”A solved problem”
  • No new classes of antibiotics for 30 years
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Stalled antibiotics development

  • ”A solved problem”
  • No new classes of antibiotics for 30 years

Walsh & Fischbach (2009) Sci. Amer.

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Stalled antibiotics development

  • ”A solved problem”
  • No new classes of antibiotics for 30 years

Accelerating resistance development

  • 25 000 deaths yearly in EU alone
  • € 1,5 billion
  • We might soon be facing a post-antibiotic era

ECDC/EMEA (2009) The bacterial challenge: time to react

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Adapted from Sandegren et al. (2012) J. Antimicrob. Chemother.

sulfonamides

”sulfa”

trimethoprim beta-lactams

pencillin, ampicillin, cefotaxime

aminoglycosides

kanamycin, streptomycin

tetracyclines copper silver arsenic macrolides

erythromycin

disinfectants

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Our target

Adapted from Sandegren et al. (2012) J. Antimicrob. Chemother.

AAC(6’) KanR

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An antibiotics adjuvant

96 8

10 20 30 40 50 60 70 80 90 100

Control sRNA MIC Kanamycin (µg/ml)

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Small RNA regulation

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Small RNA regulation

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Randomizing a sRNA library

Sharma, V, et al. (2011) ACS Synth. Biol.

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Randomization of a sRNA library

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Randomization of a sRNA library

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Screening system

Engineered sRNA library Reporter vector

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Screening system

Engineered sRNA library Reporter vector

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Screening system

Engineered sRNA library Reporter vector

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Fluorescence Activated Cell Sorter

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Fluorescence Activated Cell Sorter

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Small RNA screening

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  • Interaction with coding region of SYFP2
  • Interaction with truncated resistance mRNA

Why downregulation of fluorescence?

SD SD

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Modelling sRNA-mRNA Interactions

1 Sequencing 2 Modelling IntaRNA 3 Predictions

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Predicted binding sites

SD

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SD

Predicted binding sites

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SD

Predicted binding sites

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Clinical plasmid experiment

E.coli – MG1655 /pUUH239.2 sRNA ESBL plasmid

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Results

Downregulation of kanamycin resistance by >90%

96 12 19 8

20 40 60 80 100

Control (Native sRNA) UU17 UU37 UU55 MIC Kanamycin (µg/ml) Engineered sRNA clones

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Interaction Modeling

Binding region (predicted) Shine-Dalgarno Start Codon SD

78 % downregulation

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Interaction Modeling

Binding region (predicted) Shine-Dalgarno Start Codon

58 % downregulation

SD

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Interaction Modeling

SD Binding region (predicted) Shine-Dalgarno Start Codon

82 % downregulation

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Interaction Modeling

SD Binding region (predicted) Shine-Dalgarno Start Codon

83 % downregulation

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SD Binding region (predicted) Shine-Dalgarno Start Codon

UUconstr.

Interaction Modeling

50 % downregulation

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RBS

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RBS UUconstr.

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Common Characteristics?

Consistent hairpin formation in binding domain

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Thermodynamic Hypothesis

Adapted from Guillermo et. al., (2012), PNAS

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Under development

  • TALENs

– Transcription Activator-Like Effector Nucleases

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Under development

  • TALENs
  • Targeting gene networks
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MarR superrepressor

  • Multi Resistance

Operon

  • Non-releasing

MarR mutant (G95S)

Mutation as reported in Sulavik et al (1995) Mol. Med.

2.0 0.44

0.0 0.5 1.0 1.5 2.0 2.5 Control MarR MIC Ciprofloxacin (µg/ml)

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Delivery systems

  • Conjugative plasmid
  • Engineered phage

Lu & Collins (2009) PNAS

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Human practice

  • Blog
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Human practice

  • Blog
  • Scandinavian iGEM weekend
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Favorite parts

  • Low copy backbones

– Missing from the registry – Low copy backbones pSB4X15 – Flp recombinase sites pSB4X15(FRT) – lacIq repression pSB4X15Iq – Thermosensitive pSB8X15

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Favorite parts

  • Low copy backbones

– Missing from the registry – Low copy backbones pSB4X15 – Flp recombinase sites pSB4X15(FRT) – lacIq repression pSB4X15Iq – Thermosensitive pSB8X15

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Favorite parts

  • Low copy backbones
  • aeBlue reporter protein

Sea anemone Actinia equina

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Favorite parts

  • Low copy backbones
  • aeBlue reporter protein
  • Modular sRNA screening system

– J23101-spot42 – RFP-linker-SYFP2

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Favorite parts

  • Low copy backbones
  • aeBlue reporter protein
  • Modular sRNA screening system

– J23101-spot42 – RFP-linker-SYFP2

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The team

Supervisors Donor

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Resistance to our system?

T

  • minimize the risk of resistance development

to our system, we propose to

  • Combine two sRNA with different binding to the

mRNA

  • Target on transcriptional and translational level

– sRNA+super-repressor

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Resistance to our system?

  • Faster to develop sRNA than new antibiotics
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RFP expression by backbone

Grown overnight, IPTG (0,5 mM), Cm (12 µg/ml), triplicates (-IPTG) or quadruplicates(+IPTG).

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Plasmid yield and color development

From fluorescence +IPTG experiment.

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IPTG induction of pSB4C15Iq

Grown overnight, IPTG (0,5 mM), Cm (12 µg/ml)

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Plasmid loss at 42° C

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E-test

Spot42 control sRNA UU37