Work Report

Understanding cellular variability using droplet microfluidics

Sidhant Swarup Rout

Department of Biotechnology and Medical

Engineering

NIT Rourkela

Variation in bacterial cells

-AB

Toxin anti-toxin system

Capture cells Lyse

II I

Distribute

the lysate

III

Detect/Count

V

Amplify

IV

4

ScNAQ flowchart

Designed by: Jatin Panwar, PhD s holar, Dr. Rahul Roy’s La , Che i al E gi eeri g , IISc.

Project 1

Project 2

Project 1:Cell lysis

Lysis buffer: Mixture of BugBuster & Lysozyme

Process:

Cells cultured in LB broth

Overnight @ 37˚C

Cells pelleted

6000 rpm , 2 min

Cell lysate pelleted

6000 rpm , 2 min

Re-suspension in lysis buffer,

2 min

Re-suspension in LB broth ,

2 min

Plating in LB Agar plates (overnight

culture @ 37°C) and CFU was measured.

Optimization of Lysis buffer Concentration

3 16 5 17

500

0

100

200

300

400

500

600

0.6x B +

20KU

lysozyme

0.6x B +

40KU

lysozyme

0.6x B +

60KU

lysozyme

0.6x B +

80KU

lysozyme

0.6x B +

100KU

lysozyme

1

45

10

38

110 120

0

20

40

60

80

100

120

140

0.6x BB +

40 KU

Lysozyme

0.6x BB +

60 KU

Lysozyme

0.4x BB +

40 KU

Lysozyme

0.4x BB +

60 KU

Lysozyme

0.2x BB +

40 KU

Lysozyme

0.2x BB +

60 KU

Lysozyme

No. of colonies

Varying lysozyme concentration, Keeping

BugBuster concentration as 0.6x

Varying BugBuster concentration, Keeping

lysozyme concentration as 40 KU and 60 KU

170,5

73

0

50

100

150

200

250

0.4x BB + 60 KU

lysozyme

0.4x BB + 40 KU

lysozyme

With LB Wash

average

With LB Wash average 1

33

52

0

20

40

60

80

0.4x BB + 60 KU

lysozyme

0.4x BB + 40 KU

lysozyme

Without LB Wash

average

Without LB Wash average 2

101,75

62,5

0

20

40

60

80

100

120

140

0.4x BB + 60 KU lysozyme 0.4x BB + 40 KU lysozyme

Total average

total average

Conclusion:

Cell lysis is best achieved at a mixture of 0.4x BugBuster and 40 KU lysozyme concentration, as confirmed by

repeated experimentation.

Loop- mediated isothermal Amplification (LAMP)

• Bst DNA polymerase used

• No thermocycler required

• 6 primers used

• High product concentration

• Amplification can be achieved in shorter period of time compared to PCR

Process:

Project 2

LAMP

Ct values average obtained after optimization

of Bst DNA polymerase concentration by

evaluating LAMP reaction at to 1x, 2x and 5x

concentration..

0

10

20

30

40

50

60

JEV 10^4 copies JEV 10^3 copies No template DEN II 10^4 copies

Bst DNA polymerase Optimization

Bst Pol 1X Ct Avg Bst Pol 2X Ct Avg Bst Pol 5X Ct Avg

Gel Image:

Gel image showing negative control (no template)

amplification in case of no heat denaturation (left) and

also negative control (no template and Dengue II)

amplification in case of additional heat denaturation

(right).

Gel image showing duplicates of a LAMP reaction ,

decreasing the JEV DNA copy number from 10^5 to 1

Project 3: Nucleic acid quantification using magnetic beads

A)

B)

C)

Streptavidin magnetic beads

Biotinylated capture probe

Target DNA

Concept:

Annealing

Probe attachment

Streptavidin magnetic beads

• Magnetic beads captured inside the droplets (100 µm) (60X magnification)

Control: 1X kapa HiFi

buffer

• Magnetic beads captured inside the droplets (100 µm) (60X magnification)

Magnetic beads(1 µm)

7.3 X 10^2 beads/ µL

Microscopic detection using channels

50bp

DNA

ladder

1Kb DNA

ladder

Sample

1

Sample

2 control Gel image:

Sample 1: probe attached Template (10^6 copies) + magnetic beads (10^8 beads) – 1000 fold

dilution

Sample 2: probe attached Template (10^6 copies) + magnetic beads (10^8 beads) – 10000 fold

dilution

Control : Droplets without annealing step with biotinylated probe.

Merged droplets

*The droplets were imaged using

488 nm (blue) illumination

Future work:

• Cell Lysis: Further optimize the concentration of BugBuster and check

for efficient release of RNA.

• LAMP: Troubleshoot the contamination problem and optimize the

time of amplification. And do in vitro transcription to perform LAMP

using RNA as template.

• Droplet PCR: Try making stable droplets with magnetic beads,

perform PCR, detect and analyze data.

ACKNOWLEDGMENT

I want to thank centre of BioSystem Science and Engineering for giving this opportunity to pursue my

research interest through BioEngineering Summer Training (BEST) program.

I want to express my gratitude to Dr. Rahul Roy for his able guidance and valuable suggestions. I want

to thank Ms. Sunanda and Prof. Siddharth and Prof. Ananthasuresh for there able guidance and

support throughout the program.

I also want to thank all the research scholars in Dr. Rahul’s lab especially Jatin, Monisha, Saranya and

Priyanka for their assistance in designing and analyzing the experimental data.

Thank you!