Hok Em Sok Em RoBiotics. UMaryland igem

Transcription

1 Hok Em Sok Em RoBiotics UMaryland igem

2 Synthetic Biology is impeded by limited access to resources and public skepticism concerning downstream applications.

3 In the circuit of SynBio, there is too much resistance and too little voltage. V R

4 Increasing Voltage and Decreasing Resistance DIY thermocycler Plasmid maintenance without antibiotics Organized outreach events making UMD a center for SynBio

5 Increasing Voltage: Increasing the SynBio user base Laboratory equipment is expensive Average cost of a thermocycler: $6000 This limits access

6 Increasing Voltage: Inside the Thermocycler Advantages: Homemade Inexpensive Versatile

7 Inexpensive and Innovative Conventional Thermocycler Peltier-based Thermocycler - $6000 UMD Thermocycler Hairdryer $25 Arduino - $25 Soda can - $1 Relays - $9 Total - $60

8 Functional Cycling

9 Successful Amplification of DNA

10 Incubator The thermocycler easily doubles as an incubator. Only additional household materials are required.

11 Our thermocycler:$60 Conventional thermocycler:$6000 Savings:$5940 (99%) What can you get when your PCR only costs $60? Item 30,000 Eppendorf Tubes OR 1,000 Sequencing Reactions OR 36 kb gblocks OR 20 Miniprep kits OR 8 Jamboree Registrations OR 1 Team Registration

12 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.

13 Source of Resistance How do those directly involved confront the issue of antibiotic use? Grand View Organic Farm -No antibiotics -No GMOs -NO pesticides

14 Decreasing Resistance The Hok/Sok System: An alternative to antibiotic selection. Hok Host Killing Sok Suppression of Killing

15 Hok Sok Hok Sok

16 Hok and sok both present

17 Sok silences hok

18 Cell divides

19 Daughter cell without plasmid Daughter cell with plasmid

20 Sok degrades, hok stays Cell dies Sok still transcribed Cell lives

21 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?

22 Can Hok/Sok maintain BioBricks? CAM- CAM+ CAM- RED Plasmid without Hok/Sok PURPLE Plasmid with Hok/Sok Subculturing

23 Do cells lose plasmids when grown without a selective agent? Yes, rapidly! # Colonies 2500 Liquid Culture: No CAM, Plasmid: No Hok/Sok # hours since 1st Inoculation Trial 1 Trial 2 Trial 3 Aliquots were plated on chloramphenicol plates to assess if the plasmid had been retained.

24 # Colonies # Colonies Can Hok/Sok Maintain Plasmids? YES! Common Practice Testing Hok/Sok Liquid Culture: CAM, Plasmid: No Hok/Sok Liquid Culture: No CAM Plasmid: Hok/Sok # hours since 1st Inoculation # hours since 1st Inoculation Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3

25 OD600 Does Hok/Sok impact bacterial growth rate? No. 4 OD600 vs. Time (Minutes) Time (Minutes) Hok-Sok w/o Cam Hok-Sok w/ Cam Control w/o Cam Control w/ Cam

26 Does Hok/Sok Affect Protein Expression? RFP Hok /Sok RFP CAM+ CAM-

27 FLUORESCENCE (AU) Does Hok/Sok Affect Protein Expression? Chloramphenicol pressure reduced fluorescence over time Fluorescence vs. Time: CAM + no H/S TIME (HOURS) Trial 1 Trial 2 Trial 3

28 Fluorescence (AU) Does Hok/Sok Affect Protein Expression? Yes Hok/Sok maintained steady fluorescence 1400 Fluorescence vs. Time: CAM+ and H/S Time (Hours) Trial 1 Trial 2 Trial 3

29 Fluorescence Loss? CAM Selection (Days 1-5) Hok/Sok Selection (Days 1-6) Chloramphenicol selection leads to reduced fluorescence Cells with Hok/sok plasmid maintain fluorescence.

30 Why Does Hok/Sok Maintain Colonies without H/S, with or without chloramphenicol, acquired mutations that prevent expression. Fluorescence? Colonies with H/S did not show mutations (n=2). ATG ACG

31 Applications: Meet-up Many igem teams plan to implement their projects in the environment. High school teams benefit from homemade equipment.

32 Applications: Bioprocess Scale-Up Facility Industrial-scale production traditionally requires large amounts of antibiotic, which the hok/sok system would bypass.

33 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

34 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

35 Conclusion R V

36 Acknowledgments Kahn Lab Bentley Lab Nathan Barber Dr. Darryll J. Pines Dr. Jayanth R. Banavar Dr. Ian White Dr. Byrn B. Quimby Valerie Levanos John Wilhelm III Liz Huntley Kevin Knapstein Gary Seibel Ali Djamshidi Edward You Dr. Raymond St. Leger

37 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), doi: / (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): Department of Health and Human Services. Antimicrobials Sold Or Distributed For Use In Food-Producing Animals. Food and Drug Administration; 2013: Mitsuoki Kawano (2012) Divergently overlapping cisencoded antisense RNA regulating toxin-antitoxin systems from E. coli, RNA Biology, 9:12, , DOI: /rna.22757

38 Questions