RoBiotics UMaryland iGEM Synthetic Biology is impeded by limited - - PowerPoint PPT Presentation

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

Conventional Thermocycler

UMD Thermocycler

• Peltier-based

Thermocycler - $6000

• Hairdryer – $25
• Arduino - $25
• Soda can - $1
• Relays - $9
• Total - $60

Functional Cycling

Successful Amplification

• f DNA

Incubator

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

What can you get when your PCR only costs $60?

Our thermocycler:$60 Conventional thermocycler:$6000 Savings:$5940 (99%)

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

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

• f antibiotic use?

Grand View Organic Farm

• No antibiotics
• No GMOs
• NO pesticides

Decreasing Resistance

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

Hok Sok

Hok Sok

Hok and sok both present

Sok silences hok

Cell divides

Daughter cell with plasmid Daughter cell without plasmid

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

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?

RED – Plasmid without Hok/Sok PURPLE – Plasmid with Hok/Sok

CAM- CAM-

Subculturing

CAM+

Do cells lose plasmids when grown without a selective agent? Yes, rapidly!

500 1000 1500 2000 2500 50 100 150 200 # Colonies # hours since 1st Inoculation

Liquid Culture: No CAM, Plasmid: No Hok/Sok

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!

1000 2000 3000 4000 50 100 150 200 # Colonies # hours since 1st Inoculation

Liquid Culture: No CAM Plasmid: Hok/Sok

Trial 1 Trial 2 Trial 3 500 1000 1500 2000 2500 3000 50 100 150 200 # Colonies # hours since 1st Inoculation

Liquid Culture: CAM, Plasmid: No Hok/Sok

Trial 1 Trial 2 Trial 3

Common Practice Testing Hok/Sok

Does Hok/Sok impact bacterial growth rate? No.

0.5 1 1.5 2 2.5 3 3.5 4 100 200 300 400 500 OD600 Time (Minutes)

OD600 vs. 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 /Sok RFP RFP CAM- CAM+

Does Hok/Sok Affect Protein Expression?

Chloramphenicol pressure reduced fluorescence over time

• 500

500 1000 1500 2000 50 100 150 200 FLUORESCENCE (AU) TIME (HOURS) Fluorescence vs. Time: CAM + no H/S Trial 1 Trial 2 Trial 3

200 400 600 800 1000 1200 1400 50 100 150 200 Fluorescence (AU) Time (Hours) Fluorescence vs. Time: CAM+ and H/S Trial 1 Trial 2 Trial 3

Does Hok/Sok Affect Protein Expression? Yes

Hok/Sok maintained steady fluorescence

Fluorescence Loss?

Chloramphenicol selection leads to reduced fluorescence. Cells with Hok/sok plasmid maintain fluorescence.

Hok/Sok Selection (Days 1-6) CAM Selection (Days 1-5)

1 2 4 3 5 1 4 2 3 5 6

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

• n 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

V

R

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

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

Questions