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

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

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

4 Stalled antibiotics development A solved problem No new classes of antibiotics for 30 years Accelerating resistance development 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

5 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.

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

7 MIC Kanamycin (µg/ml) An antibiotics adjuvant Control 8 srna

8 Small RNA regulation

9 Small RNA regulation

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

11 Randomization of a srna library

12 Randomization of a srna library

13 Screening system Engineered srna library Reporter vector

14 Screening system Engineered srna library Reporter vector

15 Screening system Engineered srna library Reporter vector

16 Fluorescence Activated Cell Sorter

17 Fluorescence Activated Cell Sorter

18 Small RNA screening

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

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

21 Predicted binding sites SD

22 Predicted binding sites SD

23 Predicted binding sites SD

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

25 MIC Kanamycin (µg/ml) Results Downregulation of kanamycin resistance by >90% Control (Native srna) UU17 UU37 UU55 Engineered srna clones

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

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

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

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

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

31 RBS

32 UUconstr. RBS

33 Common Characteristics? Consistent hairpin formation in binding domain

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

35 TALENs Under development Transcription Activator-Like Effector Nucleases

36 TALENs Under development Targeting gene networks

37 MIC Ciprofloxacin (µg/ml) MarR superrepressor Multi Resistance Operon Non-releasing MarR mutant (G95S) Control MarR Mutation as reported in Sulavik et al (1995) Mol. Med.

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

39 Blog Human practice

40 Blog Human practice Scandinavian igem weekend

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

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

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

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

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

46 The team Supervisors Donor

47

48 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

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

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

51 Plasmid yield and color development From fluorescence +IPTG experiment.

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

53 Plasmid loss at 42 C

54 E-test Spot42 control srna UU37