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REU_Presentation

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REU_Presentation

  1. 1. A Robust Biosensing Platform: Refactoring the Lux Operon of V. fischeri for Maximal Bioluminescence in B. subtilis Danielle Hand Chemical Engineering
  2. 2. There is need for fast, onsite detection of chemicals in soil on a large scale Improvised Explosive Devices (IEDs) Pesticides Narcotics Detonating cord Hydrogen peroxide Urea Aluminum paste Misc. chemical precursors to explosives Heavy Metals Current narcotic detection devices are small-scale One handheld device ~$15,000 Heavy metal pollution in soil leads to water pollution Decline of pollinators Expensive uptake (vs. bacteria) Many time-consuming assays http://www.conflictarm.com/wp-content/uploads/2016/02/ Tracing_The_Supply_of_Components_Used_in_Islamic_State_IEDs.pdf
  3. 3. RBS RBS mRFP mRFP OFF ON Ligand Red fluorescence output Spectrophotometer • ~$5,000 • 4+ hours • Delicate • Relatively low resolution • Requires specific sample type Flow cytometry • ~$1.3 million • ~1 hour • High maintenance & upkeep • Cells must be dead in solution Red fluorescence in darkness Current fluorescence measurements of RNA- based biosensors do not allow easy detection Translation-based riboswitch
  4. 4. Bioluminescence luxAB Luciferase RCHO H2O + BIOLUMINESCENCE RCOOH NADPH reducing agent luxG reductase O2 RCOX + HOH --> RCOOH + XH luxD acyl-transferase RCOOH + ATP + NADPH --> NADP + AMP + PPi + RCHO luxC reductase luxE synthesase FMNH2 FMN CCD camera Current RNA-based biosensors do not allow easy detection of chemicals My solution: A robust, B. subtilis biosensing platform B. subtilis Sporulated B. subtilis Edward A. Meighan. FASEB. 7. 1016-1021. 1993.
  5. 5. Phase 1: Design and genome integration of genetically stable lux operon Operon Calculator lacI Terminators Strong constitutive promoter IPTG-inducible promoter ALPHA rational design of bacterial operons to control protein expression 1 1 3 Genetically Stable & Robust Operon luxA luxBRBS RBS luxC luxDRBS RBS luxERBS luxGRBSRBSlacIRBS Original Coding DNA Sequences (CDS) for V. fischeri from Genbank luxA luxB luxC luxD luxE luxG Genome Integration amyE ldh_1OB04_00307 Homology Arm 1 Homology Arm 2 Lux Operon B. subtilis genome Plate w/ media + potato starch Chiam Yu Ng et al. Metabolic Engineering. 29. 86-96. 2015,
  6. 6. Operon Calculator design rules Daniel Cetnar and Howard M. Salis. SEED. 2016.
  7. 7. RBS Library Calculator1.0 efficient expression optimization for multi-protein genetic systems luxA luxBRBS RBS luxC luxDRBS RBS luxERBS luxGRBS Increasing TIR (Expression) 4 G E N E T I X Colony Picker (QPix II) allows for high throughput screening to identify increased bioluminescent variants Library of lux operon variants with different levels of luminscence QuikChange Multi Site-Directed Mutagenesis of RBSs Highly bioluminescent B. subtilis Phase 2: Maximizing bioluminescence by varying protein expression levels Designed RBS libraries give a range of individual translation initiation rates Iman Farasat et al. Molecular Systems Biology. 10. 2014. 1 10 100 1000 10000 100000 0 2 4 6 8 10 12 14 16 LuxA RBS Library Sequences in library TIR AGCACATTCHAGAVGAGVTYMATTA 1 10 100 1000 10000 100000 0 2 4 6 8 10 12 14 16 LuxC RBS Library GCATCGGAMGAAAGTGASGAKGTTKWCATRGRATT 1 10 100 1000 10000 100000 1000000 0 2 4 6 8 10 12 14 16 LuxD RBS Library AAACGCGGARKTGGCDTGAGWAKGAGRTAAGAT 1 10 100 1000 10000 100000 0 2 4 6 8 10 12 14 16 LuxE RBS Library AGCACBCTCACKAAGVAGGMCACCD 1 10 100 1000 10000 100000 1000000 0 2 4 6 8 10 12 14 16 LuxG RBS Library SACAGGRAGCATACACAGAWGGNKGTAGGTM 1 10 100 1000 10000 100000 1000000 0 2 4 6 8 10 12 14 16 LuxB RBS Library GSARCGTATGGAGTTVATGACCTAWGGAKGAACYC 152,858-fold expression range 375,226-fold expression range 272,597-fold expression range 312,220-fold expression range 56,208-fold expression range 177,763-fold expression range
  8. 8. Completed objectives Genetically stable & robust operon Designed RBS Libraries

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