Hok ‘ Em Sok ‘ Em RoBiotics UMaryland iGEM
Synthetic Biology is impeded by limited access to resources and public skepticism concerning downstream applications.
In the circuit of SynBio, there is too much resistance and too little voltage. V R
Increasing Voltage and Decreasing Resistance DIY thermocycler Plasmid maintenance without antibiotics Organized outreach events making UMD a center for SynBio
Increasing Voltage: Increasing the SynBio user base • Laboratory equipment is expensive • Average cost of a thermocycler: $6000 • This limits access
Increasing Voltage: Inside the Thermocycler Advantages: • Homemade • Inexpensive • Versatile
Inexpensive and Innovative UMD Thermocycler Conventional Thermocycler • Peltier-based • Hairdryer – $25 Thermocycler - • Arduino - $25 $6000 • Soda can - $1 • Relays - $9 • Total - $60
Functional Cycling
Successful Amplification of DNA
Incubator The thermocycler easily doubles as an incubator. Only additional household materials are required.
Our thermocycler:$60 Conventional thermocycler:$6000 Savings:$5940 (99%) Item 30,000 Eppendorf Tubes OR What can you get when 1,000 Sequencing your PCR only costs Reactions OR $60? 36 kb gBlocks OR 20 Miniprep kits OR 8 Jamboree Registrations OR 1 Team Registration
Decreasing Resistance How does the public feel about the issue of antibiotic use? When we asked “ Which of the following, if any, concern you in regards to genetic engineering and biotechnology ?” 62% said “the prevalence of antibiotic use.”
Source of Resistance How do those directly involved confront the issue of antibiotic use? Grand View Organic Farm -No antibiotics -No GMOs -NO pesticides
Decreasing Resistance The Hok/Sok System: An alternative to antibiotic selection. Hok – H o st K illing Sok – S uppression o f K illing
Hok Sok Hok Sok
Hok and sok both present
Sok silences hok
Cell divides
Daughter cell with Daughter cell without plasmid plasmid
Sok degrades, hok Sok still transcribed stays Cell lives Cell dies
Can we use this natural plasmid maintenance system in synthetic biology? Does Hok/Sok: 1. effectively maintain BioBricks? 2. affect growth rate? 3. affect protein expression?
Can Hok/Sok maintain BioBricks? CAM+ CAM- CAM- RED – Plasmid without Hok/Sok PURPLE – Plasmid with Hok/Sok Subculturing
Do cells lose plasmids when grown without a selective agent? Yes, rapidly! Liquid Culture: No CAM, Plasmid: No Hok/Sok 2500 2000 # Colonies 1500 1000 500 0 0 50 100 150 200 # hours since 1st Inoculation Trial 1 Trial 2 Trial 3 Aliquots were plated on chloramphenicol plates to assess if the plasmid had been retained.
Can Hok/Sok Maintain Plasmids? YES! Common Practice Testing Hok/Sok Liquid Culture: CAM, Liquid Culture: No CAM Plasmid: No Hok/Sok Plasmid: Hok/Sok 3000 4000 2500 # Colonies # Colonies 3000 2000 1500 2000 1000 1000 500 0 0 0 50 100 150 200 0 50 100 150 200 # hours since 1st Inoculation # hours since 1st Inoculation Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3
Does Hok/Sok impact bacterial growth rate? No. OD600 vs. Time (Minutes) 4 3.5 3 2.5 OD600 2 1.5 1 0.5 0 0 100 200 300 400 500 Time (Minutes) Hok-Sok w/o Cam Control w/o Cam Hok-Sok w/ Cam Control w/ Cam
Does Hok/Sok Affect Protein Expression? Hok RFP RFP /Sok CAM+ CAM-
Does Hok/Sok Affect Protein Expression? Chloramphenicol pressure reduced fluorescence over time Fluorescence vs. Time: CAM + no H/S 2000 FLUORESCENCE (AU) 1500 1000 500 0 0 50 100 150 200 -500 TIME (HOURS) Trial 1 Trial 2 Trial 3
Does Hok/Sok Affect Protein Expression? Yes Hok/Sok maintained steady fluorescence Fluorescence vs. Time: CAM+ and H/S 1400 1200 Fluorescence (AU) 1000 800 600 400 200 0 0 50 100 150 200 Time (Hours) Trial 1 Trial 2 Trial 3
Fluorescence Loss? Hok/Sok Selection (Days 1-6) CAM Selection (Days 1-5) 2 3 5 2 3 6 1 4 1 4 5 Chloramphenicol Cells with Hok/sok selection leads to plasmid maintain reduced fluorescence. fluorescence.
Why Does Hok/Sok Maintain Fluorescence? Colonies without H/S, with or without chloramphenicol, acquired mutations that prevent expression. Colonies with H/S did not show mutations (n=2). ATG ACG
Applications: Meet-up Many iGEM teams plan to implement their projects in the environment. High school teams benefit from homemade equipment.
Applications: Bioprocess Scale-Up Facility Industrial-scale production traditionally requires large amounts of antibiotic, which the hok/sok system would bypass.
Looking Forward Hok/Sok • Couple to positive selection marker • Further examine how distance between hok/sok and second insert on plasmid affects protein expression Thermocycler • Improve consistency; optimize capacity for multiple simultaneous reactions
Conclusion • Hok/Sok: – Capable of maintaining plasmid – Does not interfere with bacterial growth – Enables alternative methods to antibiotics • Created an affordable and versatile DIY thermocycler • Meet-up and outreach efforts establish UMD as a center for SynBio collaboration
Conclusion R V
Acknowledgments • • Kahn Lab John Wilhelm III • • Bentley Lab Liz Huntley • • Nathan Barber Kevin Knapstein • • Dr. Darryll J. Pines Gary Seibel • • Dr. Jayanth R. Banavar Ali Djamshidi • • Dr. Ian White Edward You • • Dr. Byrn B. Quimby Dr. Raymond St. Leger • Valerie Levanos
References 1. Gerdes, K., Thisted, T., & Martinussen, J. (1992). Mechanism of post-segregational killing by the hok/sok system of plasmid R1: Sok antisense RNA regulates formation of a hok mRNA species correlated with killing of plasmid-free cells. Molecular Microbiology , 223(1), 1807- 1818. doi:10.1016/0022-2836(92)90714-U 2. Matsuyama T, Satoh M, Nakata R, Aoyama T, Inoue H. Functional expression of miraculin, a taste-modifying protein in Escherichia coli. J Biochem. 2009;145(4):445- 50. 3. Department of Health and Human Services. Antimicrobials Sold Or Distributed For Use In Food-Producing Animals. Food and Drug Administration; 2013:6-57. 4. Mitsuoki Kawano (2012) Divergently overlapping cis- encoded antisense RNA regulating toxin-antitoxin systems from E. coli , RNA Biology , 9:12, 1520-1527, DOI: 10.4161/rna.22757
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