1. The document describes work to enhance genetically engineered E. coli bioreporters for detecting buried landmines through their trace chemical signatures. Directed evolution was used to generate variants with improved sensitivity to 2,4-dinitrotoluene (2,4-DNT), a compound found in the vapor of TNT-based landmines. 2. Colonies were screened and the most sensitive variants to 2,4-DNT were isolated based on increased luminescence. The best variants showed a lower detection threshold of 2,4-DNT compared to the original strain. 3. For potential field use, a fluorescent reporter was developed with a simpler detection system and lower background noise than the

1. Enhancing genetically engineered
Escherichia coli bioreporters for
the detection of buried TNT-based
landmines
Eden Amiel
Supervised by: Dr. Sharon Yagur-Kroll & Prof. Shimshon Belkin
The Department of Plant and Environmental Sciences
The Institute of Life Sciences

2. This Lecture:
• 1. Introduction
• Project overview and objectives
• Landmines- background
• Implications of landmines
• Explosives trace signatures and detection techniques
• Biosensors
• Remote detection of buried landmines
• 2. Methods
• Directed Evolution
• Random Mutagenesis
• Cloning + Ligation
• Transformation
• Screening + Isolation
• 3. Results
• 4. Conclusions
• 5. Further Research

3. Project Overview
• Interdisciplinary R&D of a highly sensitive detection system
capable of covering large areas of land for the
remote detection of buried TNT-based landmines.

4. • ‘munitions placed under, or near the ground or other surface area
and designed to be exploded by the presence, or proximity of a
person or vehicle'.
Landmines - background
AT’s: pressure > 150kg
AP’s: pressure > 5kg
2,4,6-Trinitrotoluene (TNT)
-International Committee of the Red Cross, 1996

5. Implications of landmines
• Impacts on civilians safety:
1. Landmines remain dangerous after the conflict in which they were
deployed has ended, killing and injuring civilians
2. 20,000 injuries/deaths worldwide each year
3. Over half a million victims suffering from injuries caused by mines
• Impacts on the whole ecological system:
1. Access denial
2. Loss of biodiversity
3. Chemical contamination
4. Loss of productivity in arable lands
These make landmines a global problem; but despite efforts towards
landmine eradication, mines clearance remains a challenge.
1 Mine = 3$-30$ to produce, 300$-1000$ to find and clear

7. Explosives trace signatures
• Approximately 80% of all landmines are TNT-based
• These contain manufacturing impurities and degradation products
• Leakage occurs through cracks in the mine casing and vapors
diffuse through the plastic housing of the mine.
• The 3 most important vapors include:
(1) 2,4,6-TNT (2) 2,4-DNT (3) 1,3-DNB
TNT-based landmine
TNT
2,4 DNT
1,3 DNB
Surface
Gas phase
Solid phase
Underground
water
Liquid phase
Why 2,4-DNT sensor?
1. Present in the vapor phase
2. Environmentally more stable
3. Easier to work with

8. The main detection techniques
1. Metal detector
2. Chemical detection
3. Biological detection
• Dogs
• Rats
• Bees
• Plants
• Bacteria

9. Escherichia coli bioreporters
DNT-sensing element GFP-reporting element

10. Landmines and explosives biosensors
Optical
system +
detector
Immobilized sensor bacteria on soil
Beam expander
Chopper
Shutter
Laser
Mobile optical system detector

11. Remote detection of landmines
Optical systemdetector

13. Detection limits
Improvement of the sensitivity is required in order to consider
this method as applicable
‫היום‬ ‫גילוי‬ ‫טווח‬
‫רצוי‬ ‫גילוי‬ ‫טווח‬

14. How do we do it ??

15. Directed evolution
• Based on Darwinian evolution – in Nature: the survival of the fittest.
• In the lab: we generate genetic diversity in the gene of interest and
perform a powerful screening or selection assay to isolate improved
protein variants.
• Insertion of random mutations along the gene of interest (yqjF)
using non optimal PCR conditions (Error prone PCR)
• No prior knowledge of structure or function is required.
How do wegenerate genetic diversity?

16. The process
yqjF cloned up-stream
to the luxCDABE genes
1. Construct a library of
variants by error-prone PCR
2. Insert fragments into an
expression vector
3. Transform into bacteria host
4. Screen colonies with 2,4-DNT
5. Isolate improved performances
yqjF promoter
yqjF gene
KpnI SacI
KpnI SacI ‫לעמידות‬ ‫גן‬
‫לאנטיביוטיקה‬
‫הכנסת‬
‫פרומוטר‬KpnI
SacI
2
3
4
5
DH5α E.coli

17. Screen colonies with 2,4-DNT
- DNT + DNT
Divide culture into 2 plates
Grow
bacteria
over-night
Incubation, 37°C
OD & RLU measures every 20 min

18. Isolate improved promoters
• An algorithm was developed in order to find and isolate the best variants
‫האלגוריתם‬:
.1‫ערכי‬ ‫את‬ ‫שולף‬‫הלומיניסציה‬(RLU)
‫העכירות‬ ‫וערכי‬(O.D)‫קבצי‬ ‫מתוך‬
‫הגולמיות‬ ‫התוצאות‬.
.2‫ה‬ ‫מערכי‬ ‫בלאנק‬ ‫מחסר‬-O.D.
.3‫ה‬ ‫ערכי‬ ‫של‬ ‫נרמול‬ ‫מבצע‬-RLU‫ידי‬ ‫על‬
‫ה‬ ‫בערכי‬ ‫חלוקה‬-O.D.
.4‫ידי‬ ‫על‬ ‫התוצאות‬ ‫של‬ ‫אנליזה‬ ‫מבצע‬
‫והפרש‬ ‫יחס‬ ‫ערכי‬ ‫חישוב‬:
𝑰𝒏𝒅𝒖𝒄𝒕𝒊𝒐𝒏 𝑹𝒂𝒕𝒊𝒐 =
𝑹𝑳𝑼 𝑫𝑵𝑻
𝑹𝑳𝑼 𝒄𝒐𝒏𝒕𝒓𝒐𝒍
𝑰𝒏𝒅𝒖𝒄𝒕𝒊𝒐𝒏 𝑫𝒆𝒍𝒕𝒂 = 𝑹𝑳𝑼 𝑫𝑵𝑻 − 𝑹𝑳𝑼 𝒄𝒐𝒏𝒕𝒓𝒐𝒍
.5‫משני‬ ‫אחד‬ ‫לכל‬ ‫קינטיקה‬ ‫גרף‬ ‫בונה‬
‫אלו‬ ‫מחושבים‬ ‫ערכים‬,‫כל‬ ‫עבור‬
‫ווריאנט‬.‫כן‬ ‫כמו‬,‫כל‬ ‫עבור‬ ‫מחשב‬
‫גרף‬,‫מתאים‬ ‫לינארי‬ ‫קו‬.
Ratio
Time
Delta
Time
6.‫הלינארית‬ ‫המשוואה‬ ‫מתוך‬
‫לקו‬ ‫המתאימה‬,‫השיפוע‬ ‫את‬ ‫שולף‬.
7.‫השיפועים‬ ‫ערכי‬ ‫את‬ ‫מסדר‬
‫לקטן‬ ‫מהגדול‬.
8.‫עם‬ ‫הווריאנטים‬ ‫את‬ ‫כפלט‬ ‫מציג‬
‫ביותר‬ ‫הגדולים‬ ‫השיפועים‬ ‫ערכי‬
‫המדדים‬ ‫בשני‬.‫גם‬ ‫מציג‬ ‫כן‬ ‫כמו‬
‫להם‬ ‫הווריאנטים‬ ‫חמשת‬ ‫את‬
‫ה‬ ‫שיפוע‬-delta‫ביותר‬ ‫הגדול‬ ‫הינו‬
‫שני‬ ‫בחיתוך‬ ‫מופיעים‬ ‫לא‬ ‫אשר‬
‫המדדים‬.
𝑹𝑳𝑼 𝑫𝑵𝑻 < 𝑹𝑳𝑼 𝒄𝒐𝒏𝒕𝒓𝒐𝒍
?
Delta
Time
A10 + C5

19. Dose-dependent screening
Choosing colonies
with the best results
No DNT + DNT
1 2 3 4 5 6 7 8 9 10 11 12
A 100 50 25 0 100 50 25 0 100 50 25 0
B
C
D
E
F
G
H
Picking the colonies that
showed best results
Performing a dose
dependent experiment
to a select few

20. Results
0
500,000
1,000,000
1,500,000
2,000,000
2,500,000
3,000,000
0 50 100 150 200 250
Luminescence(RLU)
Time (min)
100ppm
Fwt
1st
2nd
3rd
4th-A10
4th-C5
Delta RLU = Ratio RLU =
RLU DNT
RLU control
RLU DNT − RLU control
0
10,000
20,000
30,000
40,000
50,000
60,000
70,000
80,000
90,000
0 50 100 150 200 250
Luminescence(RLU)
Time (min)
Fwt
1st
0
100,000
200,000
300,000
400,000
500,000
600,000
0 50 100 150 200 250
Luminescence(RLU)
Time (min)
Fwt
1st
2nd
0
500,000
1,000,000
1,500,000
2,000,000
2,500,000
0 50 100 150 200 250
Luminescence(RLU)
Time (min)
Fwt
1st
2nd
3rd
‫ערכי‬RLU‫אינפורמטיביים‬ ‫לא‬

21. 0
1,000
2,000
3,000
4,000
5,000
6,000
7,000
0 50 100 150 200
Luminescence(RLU)
50ppm
Time (min)
Fwt::lux
+DNT
"-DNT"
0
5,000
10,000
15,000
20,000
0 50 100 150 200
Time (min)
FB1::lux - 1st
0
100,000
200,000
300,000
400,000
500,000
600,000
0 50 100 150 200
Time (min)
FB2A1:lux - 2nd
0
500,000
1,000,000
1,500,000
2,000,000
2,500,000
0 50 100 150 200
Time (min)
FB2A1#14::lux - 3rd
0
500,000
1,000,000
1,500,000
0 50 100 150 200
Time (min)
F-A10::lux - 4th
0
500,000
1,000,000
1,500,000
2,000,000
0 50 100 150 200
Time (min)
F-C5::lux - 4th
0
5,000
10,000
15,000
20,000
25,000
30,000
35,000
40,000
45,000
50,000
0 50 100 150 200
Luminescence(RLU)
50ppm
Time (min)
FB2A1#14::lux - 3rd

22. ΔRLU
0
100
200
300
400
500
600
700
800
0 20 40 60 80 100
MaxRatioover300min
DNT mg/l
MG/Fwt
1st
2nd
3rd
4th-A10
4th-C5
Ratio RLU
0
500,000
1,000,000
1,500,000
2,000,000
2,500,000
3,000,000
0 20 40 60 80 100
Max∆RLUover300min
DNT mg/l
MG/Fwt
1st
2nd
3rd
4th-A10
4th-C5

23. ΔRLU and Ratio at 25ppm
0
50
100
150
200
250
300
350
400
0 50 100 150 200 250 300 350
Ratio
25ppm
Time (min)
Fwt
1st
2nd
3rd
4th-A10
4th-C5
0
500,000
1,000,000
1,500,000
2,000,000
2,500,000
0 50 100 150 200 250 300 350
∆RLU
25ppm
Time (min)
Fwt
1st
2nd
3rd
4th-A10
4th-C5

24. Detection threshold
0
5
10
15
20
25
30
Fwt 1st 2nd 3rd 4th-A10 4th-C5
Sensitivitymg/l(EC200)
EC200 = the concentration which induces a response (Ratio = 2)

25. Transfer to a system suitable
for field measuring
• Luminescence: Using luxCDABE genes,
stronger responses, simpler to use,
less background noise.
• Fluorescence: Using GFP gene, a more
specific response suitable for the field
detection system
DNT-sensing element GFP-reporting elementluxCDABE-reporting element

26. 0
2,000
4,000
6,000
8,000
10,000
12,000
14,000
16,000
18,000
0 20 40 60 80 100
Max∆RFUover300min
DNT mg/l
MG/Fwt
1st
2nd
3rd
4th-A10
4th-C5
0
1
2
3
4
5
6
0 20 40 60 80 100
MaxRatioover300min
DNT mg/l
MG/Fwt
1st
2nd
3rd
4th-A10
4th-C5
ΔRFU and Ratio RFU

27. ΔRFU and Ratio at 25ppm
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0 50 100 150 200 250 300 350
Ratio
25ppm
Time
Fwt
1st
2nd
3rd
4th-A10
4th-C5
0
1000
2000
3000
4000
5000
6000
7000
8000
0 50 100 150 200 250 300 350
ΔRFU
25ppm
Time
Fwt
1st
2nd
3rd
4th-A10
4th-C5

28. 0
20
40
60
80
100
120
Fwt 1st 2nd 3rd 4th-A10 4th-C5
Sensitivitymg/l(EC200)
Detection threshold
EC200 = the concentration which induces a response (Ratio = 2)

29. WT CGGTTTTGGCGTATGGAGCGCCTGGCGTCTGGTTAAAACGACCCTCAAGCAGCAACAGCTTCGCGGTTAA
FB2 ..........................A...........................................
FB2A1 ..........................A...........................................
FB2A1#14 ..........................A...............T..........................G
4th-A10 ..........................A...............T..........................G
4th-C5 ..........................A...............T.................C........G
WT CTTCCCTCTGGCCGGAGCCATTCCGGCCTTATCCCTCAAATTTTTTGAAGATTTTTGACAGTTTTCCTTG
FB2 ............................................................A.........
FB2A1 ....................................................C.......A.........
FB2A1#14 ...............................................G....C....T..A.........
4th-A10 ...............................................G....C....T..A.........
4th-C5 ...............................................G....C....T..A.........
WT CTAACAATCATCATTCACCACGTTTATGATTCTCTCCATCGACAGCAACGACGCTAATACCGCGCCATTG
FB2 ......................................................................
FB2A1 ..................................................................T...
FB2A1#14 ..................................................................T...
4th-A10 ..................................................................TC..
4th-C5 .....................A......................A.........C...........T...
-35
-10
+1
F26 F36
F49
F48F3
F124F128F133F138
F186
F195
F213F230
yqjF promoter

30. Point Mutations – Individual effects
0
200
400
600
800
1,000
1,200
1,400
1,600
1,800
2,000
0 20 40 60 80 100 120
MaxRatioover240'
2,4-DNT mg/l
MG/Fwt::lux
MG/F48::lux
MG/F49::lux
MG/F36:lux
MG/F26::lux
MG/F3::lux
MG/F124::lux
MG/F128:lux
MG/F133::lux
MG/F138::lux
MG/F186::lux
MG/F195::lux
MG/F213::lux
MG/F230::lux
MG/F138:lux

31. Point Mutations – Individual
-35 region of σ70
115.1
611.9
1612.0
1774.8
14.5 22.9 40.2 49.4
100.7
0.0
200.0
400.0
600.0
800.0
1000.0
1200.0
1400.0
1600.0
1800.0
2000.0
Fwt::lux F124 F128 F133 F138 F3 F26 F48 F49
ratioat100mg/l
‫ב‬ ‫שנכנסה‬ ‫היחידה‬ ‫המוטציה‬-A10
‫לבד‬:‫אפקט‬ ‫אין‬
‫נוספות‬ ‫מוטציות‬ ‫עם‬ ‫בשילוב‬:‫שיפור‬ ‫יש‬

32. Point Mutations – Multiple
0
500,000
1,000,000
1,500,000
2,000,000
2,500,000
3,000,000
3,500,000
4,000,000
4,500,000
0 20 40 60 80 100 120
MaxDeltaRLUover240'
2,4-DNT mg/l
MG/Fwt::lux
MG/FB2::lux
MG/FB2A1::lux
MG/FB2A1::lux#14
MG/F124-133:lux
MG/F124-138:lux
MG/F124,128::lux
MG/F124,133::lux
MG/F128,133::lux
MG/F213::lux
MG/F230::lux
MG/F138:lux
0
500,000
1,000,000
1,500,000
2,000,000
2,500,000
3,000,000
3,500,000
4,000,000
4,500,000
0 20 40 60 80 100 120
MaxDeltaRLUover240'
2,4-DNT mg/l
MG/Fwt::lux
MG/FB2A1::lux#14
MG/F124-133:lux
MG/F124-138:lux
MG/F213::lux
MG/F138:lux
0
5,000
10,000
15,000
20,000
25,000
30,000
0 20 40 60 80 100 120
MaxDeltaRLUover240'
2,4-DNT mg/l
MG/Fwt::lux
MG/F213::lux
MG/F138:lux
13
𝑘
=
13!
𝑘! 13−𝑘 !
13
7
=
13!
7! 13−7 !
1716 Possible combinations
Very low Ratio = 2
504 535
3,103
15,636
6,634
3,219 2,054
124,008
770 420 528
1
10
100
1,000
10,000
100,000
1,000,000
RLU
‫פי‬ ‫גבוה‬ ‫רקע‬250‫מה‬-WT!

33. Detection threshold
EC200 = the concentration which induces a response (Ratio = 2)
0
5
10
15
20
25
30
35
40
MG/Fwt::lux FB2 FB2A1 FB2A1#14 F124-133 F124-138
Sensitivitymg/l(EC200)
‫המוטציה‬F138‫הלומינסנציה‬ ‫פעילות‬ ‫את‬ ‫מורידה‬ ‫אמנם‬
‫המערכת‬ ‫של‬ ‫הרגישות‬ ‫סף‬ ‫את‬ ‫מעלה‬ ‫אך‬

34. Conclusions
• A10 + C5 have both high Ratio and high Delta
whereas FB2A1#14 has high Delta but low Ratio and
FB2A1 has high Ratio but low Delta
• Some specific mutations lower the performance of
the system but might increase performance when
combined with other mutations.

35. Further Research
• Performing RM to a specific area in the promoter,
such as the -35 domain
• Searching for TF binding sites and planning
mutations accordingly
• Applying different approaches to further lower the
detection threshold

36. • Using mutant strains of several membrane proteins as hosts
for the genetic fusion
• Increasing the influx levels of the substance through the
fusion of the membrane protein OmpF porin gene to an IPTG-
inducible lacZ gene promoter.
Different approaches to lower the
detection threshold
Δ
2,4-DNT

37. • Increasing the influx levels of the substance through the
fusion of the membrane protein OmpF porin gene to an IPTG-
inducible lacZ gene promoter.
Different approaches to lower the
detection threshold
2,4-DNT
ompF
OmpF porin

38. Thanks
• Prof. Shimshon Belkin
• Dr. Sharon Yagur-Kroll
• Lab Team:
• Dr. Rachel Rosen
• Dr. Tal Elad
• Dr. Keren Harel-Dasa
• Omri Finkel
• Neta Bachar
• Bini Shemer
• Noa Palevski
• Adi Fainshtain
• Yaara Moskovitz