UC Davis 2013
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iGEM 2013 UC Davis RiboTALs
Synthetic Biology
INPUT OUTPUT
Controlled by Transcription Factors
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iGEM 2013 UC Davis RiboTALs
Need for Input Diversity
• Controlled by Transcription Factors
5 5n
INPUTS OUTPUTS
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iGEM 2013 UC Davis RiboTALs
Sequence Specific
Easily Engineerable Target Specificity
Tunable Output Levels
Ability to Multiplex
Variable Input
Usable in any Chassis
Design Criteria for Ideal
Transcription Factors
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iGEM 2013 UC Davis RiboTALs
Transcription Factors
Sequence Specific
Easily Engineerable Target Specificity
Tunable Output Levels
Ability to Multiplex
Variable Input
Usable in any Chassis
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iGEM 2013 UC Davis RiboTALs
Designing Novel
Transcription Factors
DNA
RNA
Protein 6
iGEM 2013 UC Davis RiboTALs
TALEs
As a Transcription Factor:
Sequence Specific
Easily Engineerable
Target Specificity
Tunable
Usable in any Chassis
DNA
RNA
E. Pennisi, "The tale of the TALEs," in Science. vol. 338, ed United States, 2012, pp. 1408-11.
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iGEM 2013 UC Davis RiboTALs
Designing Novel
Transcription Factors
DNA
RNA
Protein 8
iGEM 2013 UC Davis RiboTALs
RNA
Riboswitches
Riboswitches Attached to
Transcription Factors:
Tunable Output
Levels
Ability to Multiplex
Variable Input
A. Serganov and E. Nudler, "A Decade of Riboswitches," Cell, vol. 152, pp. 17-24, 1/17/ 2013.
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+ inducer
Protein
iGEM 2013 UC Davis RiboTALs
Design Criteria for
IdealTranscription Factors Typical
Transcription
Factor
RiboTALes
Sequence Specific Easily Engineerable
Target Specificity
Tunable Output Levels
Ability to Multiplex
Variable Input Usable in any Chassis
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iGEM 2013 UC Davis RiboTALs
Building Our Testing
Constructs
iGEM 2013 UC Davis RiboTALs
Building Our Testing
Constructs
iGEM 2013 UC Davis RiboTALs
Testing Construct
TALe
pBAD
GFP
pTET TBS
+ aTc
iGEM 2013 UC Davis RiboTALs
Testing Construct
TALe
pBAD
GFP
pTET TBS
+ aTc
+ arabinose
TALe
Riboswitch
iGEM 2013 UC Davis RiboTALs
Testing Construct
TALe
pBAD
GFP
pTET TBS
+ aTc
+ arabinose
TALe
Riboswitch
+ theophylline
TALe
TALe
iGEM 2013 UC Davis RiboTALs
Testing Construct
iGEM 2013 UC Davis RiboTALs
Experiments
• Host Organism: E. coli MG1655Z1
• Runs extended into cellular stationary phase
• Equipment: o TECAN Infinite 200Pro Microplate Reader
o 96 well plates
o Awesome pipetting skills
iGEM 2013 UC Davis RiboTALs
Experiment 1:
• Subject RiboTALe to a theophylline gradient
TALe
Riboswitch
+ theophylline
TALe
iGEM 2013 UC Davis RiboTALs
Translation is modulated
by theophylline
iGEM 2013 UC Davis RiboTALs
Experiment 2:
• Compare effect of TALe binding affinities on
system response
GFP
pTET TBS2
GFP
pTET TBS
TALe Kd = X
TALe Kd = Y
iGEM 2013 UC Davis RiboTALs
TAL repressor binding
affinities provide tunability
iGEM 2013 UC Davis RiboTALs
Experiment 3:
• Compare effect of riboswitch leakiness on
system response
TALe
Riboswitch
TALe
Riboswitch
iGEM 2013 UC Davis RiboTALs
Riboswitch leakiness
modulates activity
iGEM 2013 UC Davis RiboTALs
Experiment 4:
• Full characterization of RiboTALe device
TALe
pBAD
GFP
pTET TBS
+ aTc
+ arabinose
TALe Riboswitch
+ theophylline
TALe
iGEM 2013 UC Davis RiboTALs
3D RiboTALe Data
Characterization
25 With lots of data, what is the best way to share it?
iGEM 2013 UC Davis RiboTALs
Modeling
• Mathematical support
for device functionality.
• Can be developed into
a predictive tool for
RiboTALe design
iGEM 2013 UC Davis Human Outreach
Raw Data for RiboTALs
• Complex system with
multiple inducers
• How can we best share?
iGEM 2013 UC Davis Human Outreach
iGEM 2013 UC Davis RiboTALs
Scaling to All Parts
iGEM 2013UC Davis Human Outreach
Initial Population
Bielefeld-Germany
ZJU-China
Stanford-Brown
UCSF
iGEM 2013 UC Davis Human Outreach
A Walkthrough: Uploading
iGEM 2013 UC Davis Human Outreach
A Walkthrough: Searching
iGEM 2013 UC Davis Human Outreach
A Walkthrough: Results
iGEM 2013 UC Davis Human Outreach
• After Downloading
Data:
• Able to Visualize
Output from Data Depot
iGEM 2013 UC Davis Human Outreach
iGEM 2013 UC Davis Human Outreach
Other Efforts in
Human Outreach
• Provided lab materials for a
biotechnology class at
Davis Senior High School.
• Worked with Nicholas
Armstrong to create a
Synthetic Biology Club at
UC Davis o Platform for new student
research
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iGEM 2013 UC Davis RiboTALs
Accomplishments and
Future Directions
• Introduced a novel set of transcription factors
known as RiboTALs. • New riboswitch and TALe combinations
• Use modeling to predict and tune device behavior
• Developed the Depot for raw data
characterization of Biobricks.
• Further develop the Depot
• Expand the amount of data in the Depot
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iGEM 2013 UC Davis RiboTALs
Acknowledgements
Our Advisers:
• Dr. Marc Facciotti
• Dr. IliasTagkopoulos
• Dr. Justin B. Siegel
Technical Guidance:
• Andrew Yao
• Dr. NavneetRai
• Linh Nguyen
Special Thanks to:
• Nick Csicsery
• Dr. Justin B. Gallivan
• Dr. YoheiYokobayashi
• Dr. Bertram Ludaescher
• Sven Koehler
• AnandarupSarkar
• Hector Garcia Martin
• Keegan Owsley
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iGEM 2013 UC Davis RiboTALs
References 1. E. Pennisi, "The tale of the TALEs," in Science. vol. 338, ed United States, 2012, pp.
1408-11.
2. A. Serganov and E. Nudler, "A Decade of Riboswitches," Cell, vol. 152, pp. 17-24,
1/17/ 2013.
3. J. F. Meckler, M. S. Bhakta, M. S. Kim, R. Ovadia, C. H. Habrian, A. Zykovich, et al.,
"Quantitative analysis of TALE-DNA interactions suggests polarity effects," Nucleic
Acids Res, vol. 41, pp. 4118-28, Apr 2013.
4. S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic
riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.
5. S. Topp, C. M. K. Reynoso, J. C. Seeliger, I. S. Goldlust, S. K. Desai, D. Murat, et al.,
"Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species
(vol 76, pg 7881, 2010)," Applied and Environmental Microbiology, vol. 77, pp. 2199-
2199, Mar 2011.
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iGEM 2013 UC Davis RiboTALs
Thank You
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