1. A Robust Biosensing Platform:
Refactoring the Lux Operon of V. fischeri
for Maximal Bioluminescence in B. subtilis
Danielle Hand
Chemical Engineering

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. 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. 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. 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. Operon Calculator design rules
Daniel Cetnar and Howard M. Salis. SEED. 2016.

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. Completed objectives
Genetically stable & robust operon Designed RBS Libraries