Work Report. Understanding cellular variability using droplet microfluidics
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1 Work Report Understanding cellular variability using droplet microfluidics Sidhant Swarup Rout Department of Biotechnology and Medical Engineering NIT Rourkela
2 Variation in bacterial cells -AB
3 Toxin anti-toxin system
4 ScNAQ flowchart II I Capture cells Project 1 Lyse III Distribute the lysate Detect/Count V Project 2 Amplify IV Designed by: Jatin Panwar, PhD s holar, Dr. Rahul Roy s La, Che i al E gi eeri g, IISc. 4
5 Project 1:Cell lysis Lysis buffer: Mixture of BugBuster & Lysozyme
6 Process: Cells cultured in LB broth 37 C Cells pelleted 6000 rpm, 2 min Re-suspension in lysis buffer, 2 min Plating in LB Agar plates (overnight 37 C) and CFU was measured. Cell lysate pelleted 6000 rpm, 2 min Re-suspension in LB broth, 2 min
7 Optimization of Lysis buffer Concentration x B + 0.6x B + 0.6x B + 0.6x B + 0.6x B + 20KU 40KU 60KU 80KU 100KU lysozyme lysozyme lysozyme lysozyme lysozyme x BB + 0.6x BB + 40 KU 60 KU Lysozyme Lysozyme 0.4x BB + 0.4x BB + 40 KU 60 KU Lysozyme Lysozyme 0.2x BB + 0.2x BB + 40 KU 60 KU Lysozyme 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
8 Without LB Wash average With LB Wash average x BB + 60 KU lysozyme 170, x BB + 60 KU lysozyme 0.4x BB + 40 KU lysozyme 0.4x BB + 40 KU lysozyme With LB Wash average 1 Without LB Wash average 2 Total average ,75 62,5 0.4x BB + 60 KU lysozyme 0.4x BB + 40 KU lysozyme 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.
9 Project 2 Loop- mediated isothermal Amplification (LAMP) Process: 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
10 LAMP Bst DNA polymerase Optimization 60 Ct values average obtained after optimization of Bst DNA polymerase concentration by evaluating LAMP reaction at to 1x, 2x and 5x concentration JEV 10^4 copies Bst Pol 1X Ct Avg JEV 10^3 copies Bst Pol 2X Ct Avg No template DEN II 10^4 copies Bst Pol 5X Ct Avg
11 Gel Image: Gel image showing duplicates of a LAMP reaction, decreasing the JEV DNA copy number from 10^5 to 1 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).
12 Project 3: Nucleic acid quantification using magnetic beads
13 Concept: A) Annealing B) Probe attachment C) Streptavidin magnetic beads Biotinylated capture probe Target DNA
14 Streptavidin magnetic beads Magnetic beads captured inside the droplets (100 µm) (60X magnification) Control: 1X kapa HiFi buffer Magnetic beads(1 µm) 7.3 X 10^2 beads/ µl
15 Microscopic detection using channels Merged droplets *The droplets were imaged using 488 nm (blue) illumination Gel image: 50bp DNA ladder Sample 1 Sample 2 control 1Kb DNA ladder 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) fold dilution Control : Droplets without annealing step with biotinylated probe.
16 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.
17 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.
18 Thank you!
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