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

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

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

macrolides erythromycin beta-lactams pencillin, ampicillin, cefotaxime sulfonamides sulfa aminoglycosides kanamycin, streptomycin trimethoprim tetracyclines disinfectants silver copper arsenic Adapted from Sandegren et al. (2012) J. Antimicrob. Chemother.

AAC(6 ) Kan R Our target Adapted from Sandegren et al. (2012) J. Antimicrob. Chemother.

MIC Kanamycin (µg/ml) An antibiotics adjuvant 100 90 80 70 60 50 40 30 20 10 0 96 Control 8 srna

Small RNA regulation

Randomizing a srna library Sharma, V, et al. (2011) ACS Synth. Biol.

Randomization of a srna library

Screening system Engineered srna library Reporter vector

Fluorescence Activated Cell Sorter

Small RNA screening

Why downregulation of fluorescence? Interaction with coding region of SYFP2 SD Interaction with truncated resistance mrna SD

Modelling srna-mrna Interactions 1 Sequencing 2 Modelling IntaRNA 3 Predictions

Predicted binding sites SD

Clinical plasmid experiment srna ESBL plasmid E.coli MG1655 /puuh239.2

MIC Kanamycin (µg/ml) Results Downregulation of kanamycin resistance by >90% 100 96 80 60 40 20 0 Control (Native srna) 19 12 8 UU17 UU37 UU55 Engineered srna clones

Interaction Modeling SD 78 % downregulation Shine-Dalgarno Start Codon Binding region (predicted)

Interaction Modeling SD 58 % downregulation Shine-Dalgarno Start Codon Binding region (predicted)

Interaction Modeling SD 82 % downregulation Shine-Dalgarno Start Codon Binding region (predicted)

Interaction Modeling SD 83 % downregulation Shine-Dalgarno Start Codon Binding region (predicted)

Interaction Modeling SD UUconstr. 50 % downregulation Shine-Dalgarno Start Codon Binding region (predicted)

RBS

UUconstr. RBS

Common Characteristics? Consistent hairpin formation in binding domain

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

TALENs Under development Transcription Activator-Like Effector Nucleases

TALENs Under development Targeting gene networks

MIC Ciprofloxacin (µg/ml) MarR superrepressor Multi Resistance Operon 2.5 2.0 2.0 Non-releasing MarR mutant (G95S) 1.5 1.0 0.5 0.44 0.0 Control MarR Mutation as reported in Sulavik et al (1995) Mol. Med.

Conjugative plasmid Engineered phage Delivery systems Lu & Collins (2009) PNAS

Blog Human practice

Blog Human practice Scandinavian igem weekend

Favorite parts Low copy backbones Missing from the registry Low copy backbones psb4x15 Flp recombinase sites psb4x15(frt) laciq repression psb4x15iq Thermosensitive psb8x15

Favorite parts Low copy backbones aeblue reporter protein Sea anemone Actinia equina

Favorite parts Low copy backbones aeblue reporter protein Modular srna screening system J23101-spot42 RFP-linker-SYFP2

The team Supervisors Donor

Resistance to our system? To 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

Resistance to our system? Faster to develop srna than new antibiotics

RFP expression by backbone Grown overnight, IPTG (0,5 mm), Cm (12 µg/ml), triplicates (-IPTG) or quadruplicates(+iptg).

Plasmid yield and color development From fluorescence +IPTG experiment.

IPTG induction of psb4c15iq Grown overnight, IPTG (0,5 mm), Cm (12 µg/ml)

Plasmid loss at 42 C

E-test Spot42 control srna UU37