Enhancing genetically engineered Escherichia coli bioreporters forthe detection of buried TNT-based landmines

Eden AmielSupervised by: Dr. Sharon Yagur-Kroll & Prof. Shimshon BelkinThe Department of Plant and Environmental SciencesThe Institute of Life Sciences

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

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.

• ‘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

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 year3. Over half a million victims suffering from injuries caused by mines

• Impacts on the whole ecological system:1. Access denial2. Loss of biodiversity3. Chemical contamination4. 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

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

TNT2,4 DNT1,3 DNB

Surface

Gas phase

Solid phase

Underground water

Liquid phase

Why 2,4-DNT sensor?1. Present in the vapor phase2. Environmentally more stable 3. Easier to work with

The main detection techniques

1. Metal detector 2. Chemical detection 3. Biological detection

• Dogs • Rats• Bees• Plants• Bacteria

Escherichia coli bioreporters

DNT-sensing element GFP-reporting elementyqjF gene promoter

Landmines and explosives biosensors

Optical system + detector

Immobilized sensor bacteria on soil

Telescope

Beam expander

Chopper

ShutterLaser

Mobile optical system detector

330

mW

laser beam at λ=445nm

Remote detection of landminesOptical systemdetector

Detection limits

Improvement of the sensitivity is required in order to consider this method as applicable

היום גילוי טווח

רצוי גילוי טווח

How do we do it ??

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 we generate genetic diversity?

The process

yqjF cloned up-streamto 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 geneKpnI SacI

KpnI SacI לעמידות גןלאנטיביוטי

קה

הכנסת KpnIפרומוטר

SacI

2

3

4

5

X4DH5α E.coli

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

Isolate improved promoters• An algorithm was developed in order to find and isolate the best variants

האלגוריתם:הלומיניסציה 1. ערכי את שולף

(RLU( )העכירות מתוך( O.Dוערכי. הגולמיות התוצאות קבצי

.2- ה מערכי בלאנק .O.Dמחסר

.3- ה ערכי של נרמול על RLUמבצע- ה בערכי חלוקה .O.Dידי

על 4. התוצאות של אנליזה מבצע: והפרש יחס ערכי חישוב ידי

אחד 5. לכל קינטיקה גרף בונה , עבור אלו מחושבים ערכים משני , . עבור מחשב כן כמו ווריאנט כל

. , מתאים לינארי קו גרף כל

Time

Ratio

Time

Delta

הלינארית. 6 המשוואה מתוך , את שולף לקו המתאימה

השיפוע.השיפועים. 7 ערכי את מסדר

. לקטן מהגדולהווריאנטים. 8 את כפלט מציג

הגדולים השיפועים ערכי עם . כן כמו המדדים בשני ביותר

חמשת את גם מציגשיפוע להם הווריאנטים

אשר deltaה- ביותר הגדול הינושני בחיתוך מופיעים לא

המדדים.

𝑹𝑳𝑼 (𝑫𝑵𝑻 )<𝑹𝑳𝑼 (𝒄𝒐𝒏𝒕𝒓𝒐𝒍 )?

Time

Delta

A10 + C5

Dose-dependent screening

Choosing colonieswith the best results

No DNT + DNT

1 2 3 4 5 6 7 8 9 10 11 12A 100 50 25 0 100 50 25 0 100 50 25 0BCDEFGH

Picking the colonies that showed best results

Performing a dose dependent experiment to a select few

Results

0 50 100 150 200 2500

500,000

1,000,000

1,500,000

2,000,000

2,500,000

3,000,000

100ppm

Fwt1st2nd3rd4th-A104th-C5

Time (min)

Lum

ines

cenc

e (R

LU)

Delta RLU = Ratio RLU =RLU (DNT )RLU (control )

RLU (DNT )− RLU (control )

0 50 100 150 200 2500

10,000

20,000

30,000

40,000

50,000

60,000

70,000

80,000

90,000

Fwt1st

Time (min)

Lum

ines

cenc

e (R

LU)

0 50 100 150 200 2500

100,000

200,000

300,000

400,000

500,000

600,000

Fwt1st2nd

Time (min)

Lum

ines

cenc

e (R

LU)

0 50 100 150 200 2500

500,000

1,000,000

1,500,000

2,000,000

2,500,000

Fwt1st2nd3rd

Time (min)

Lum

ines

cenc

e (R

LU)

אינפורמטיביים RLUערכי לא

0 20 40 60 80 100 120 140 160 180 2000

1,0002,0003,0004,0005,0006,0007,000

Fwt::lux

+DNT"-DNT"

Time (min)

Lum

ines

cenc

e (R

LU)

50pp

m

0 20 40 60 80 100 120 140 160 180 2000

5,000

10,000

15,000

20,000

FB1::lux - 1st

Time (min)

0 20 40 60 80 100 120 140 160 180 2000

100,000200,000300,000400,000500,000600,000

FB2A1:lux - 2nd

Time (min) 0 20 40 60 80 100 120 140 160 180 2000

500,000

1,000,000

1,500,000

2,000,000

2,500,000

FB2A1#14::lux - 3rd

Time (min)

0 20 40 60 80 100 120 140 160 180 2000

200,000400,000600,000800,000

1,000,0001,200,0001,400,000

F-A10::lux - 4th

Time (min)

0 20 40 60 80 100 120 140 160 180 2000

500,0001,000,0001,500,0002,000,000

F-C5::lux - 4th

Time (min)

0 20 40 60 80 100 120 140 160 180 2000

5,000

10,000

15,000

20,000

25,000

30,000

35,000

40,000

45,000

50,000

FB2A1#14::lux - 3rd

Time (min)

Lum

ines

cenc

e (R

LU)

50pp

m

ΔRLU

0 10 20 30 40 50 60 70 80 90 1000

100

200

300

400

500

600

700

800

MG/Fwt1st2nd3rd4th-A104th-C5

DNT mg/l

Max

Rati

o ov

er 3

00 m

in

Ratio RLU

0 10 20 30 40 50 60 70 80 90 1000

500,000

1,000,000

1,500,000

2,000,000

2,500,000

3,000,000

MG/Fwt1st2nd3rd4th-A104th-C5

DNT mg/l

Max

∆RL

U ov

er 3

00 m

in

ΔRLU and Ratio at 25ppm

0 50 100 150 200 250 300 3500

50

100

150

200

250

300

350

400

Fwt1st2nd3rd4th-A104th-C5

Time (min)

Ratio

25pp

m

0 50 100 150 200 250 300 3500

500,000

1,000,000

1,500,000

2,000,000

2,500,000

Fwt1st2nd3rd4th-A104th-C5

Time (min)

∆RLU

25pp

m

Detection threshold

Fwt 1st 2nd 3rd 4th-A10 4th-C50

2

4

6

8

10

12

14

16

18

20

Sens

itivi

ty m

g/l (

EC20

0)

EC200 = the concentration which induces a response (Ratio = 2)

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

0 10 20 30 40 50 60 70 80 90 1000

2,0004,0006,0008,000

10,00012,00014,00016,00018,000

MG/Fwt1st2nd3rd4th-A104th-C5

DNT mg/l

Max

∆RF

U ov

er 3

00 m

in

0 10 20 30 40 50 60 70 80 90 1000

1

2

3

4

5

6

MG/Fwt1st2nd3rd4th-A104th-C5

DNT mg/l

Max

Rati

o ov

er 3

00 m

in

ΔRFU and Ratio RFU

ΔRFU and Ratio at 25ppm

0 50 100 150 200 250 300 3500.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Fwt1st2nd3rd4th-A104th-C5

Time

Ratio

25pp

m

0 50 100 150 200 250 300 3500

1000

2000

3000

4000

5000

6000

7000

8000

Fwt1st2nd3rd4th-A104th-C5

Time

ΔRFU

25pp

m

Fwt 1st 2nd 3rd 4th-A10 4th-C50

102030405060708090

100

Sens

itivi

ty m

g/l (

EC20

0)Detection threshold

EC200 = the concentration which induces a response (Ratio = 2)

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

Point Mutations – Individual effects

0 20 40 60 80 100 1200

200400600800

1,0001,2001,4001,6001,8002,000 MG/Fwt::lux

MG/F48::luxMG/F49::luxMG/F36:luxMG/F26::luxMG/F3::luxMG/F124::luxMG/F128:luxMG/F133::luxMG/F138::luxMG/F186::luxMG/F195::luxMG/F213::luxMG/F230::luxMG/F138:lux

2,4-DNT mg/l

Max

Rati

o ov

er 2

40'

Point Mutations – Individual

-35 region of σ70

Fwt::lux F124 F128 F133 F138 F3 F26 F48 F490.0

200.0

400.0

600.0

800.0

1000.0

1200.0

1400.0

1600.0

1800.0

2000.0

115.1

611.9

1612.0

1774.8

14.5 22.9 40.2 49.4100.7

Chart Title

ratio

at 1

00 m

g/l

- ב שנכנסה היחידה A10המוטציהאפקט: לבד אין

נוספות מוטציות עם שיפור: בשילוב יש

Point Mutations – Multiple

0 20 40 60 80 100 1200

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 MG/Fwt::luxMG/FB2::luxMG/FB2A1::luxMG/FB2A1::lux#14MG/F124-133:luxMG/F124-138:luxMG/F124,128::luxMG/F124,133::luxMG/F128,133::luxMG/F213::luxMG/F230::luxMG/F138:lux

2,4-DNT mg/l

Max

Del

ta R

LU o

ver 2

40'

0 20 40 60 80 100 1200

500,000

1,000,000

1,500,000

2,000,000

2,500,000

3,000,000

3,500,000

4,000,000

4,500,000MG/Fwt::lux

MG/FB2A1::lux#14

MG/F124-133:lux

MG/F124-138:lux

MG/F213::lux

MG/F138:lux

2,4-DNT mg/l

Max

Del

ta R

LU o

ver 2

40'

0 20 40 60 80 100 1200

5,000

10,000

15,000

20,000

25,000

30,000

MG/Fwt::lux

MG/F213::lux

MG/F138:lux

2,4-DNT mg/l

Max

Del

ta R

LU o

ver 2

40'

= = 1716 Possible combinations

Very low Ratio

Fwt 1st 2nd 3rd F124 F128 F133 F124-133

F137 F138 F145100

1,000

10,000

100,000

1,000,000

504 535

3,103

15,6366,634

3,219 2,054

124,008

770420 528

RLU פי גבוה -250רקע !WTמה

Detection thresholdEC200 = the concentration which induces a response (Ratio = 2)

MG/Fwt::lux

FB2 FB2A1 FB2A1#14 F124-133 F124-1380

2

4

6

8

10

12

Sens

itivi

ty m

g/l (

EC20

0)

הלומינסנציה F138המוטציה פעילות את מורידה אמנםהמערכת של הרגישות סף את מעלה אך

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.

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

• 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

• 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

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

Questions