UC Davis 20132013.igem.org/files/presentation/UC_Davis.pdf · iGEM 2013 UC Davis RiboTALs TALEs As a Transcription Factor: Sequence Specific Easily Engineerable Target Specificity

UC Davis 2013

iGEM 2013 UC Davis RiboTALs

Synthetic Biology

INPUT OUTPUT

Controlled by Transcription Factors

Need for Input Diversity

• Controlled by Transcription Factors

INPUTS OUTPUTS

Sequence Specific

Easily Engineerable Target Specificity

Tunable Output Levels

Ability to Multiplex

Variable Input

Usable in any Chassis

Design Criteria for Ideal

Transcription Factors

Sequence Specific

Easily Engineerable Target Specificity

Variable Input

Designing Novel

Protein 6

As a Transcription Factor:

Sequence Specific

Easily Engineerable

Target Specificity

Tunable

E. Pennisi, "The tale of the TALEs," in Science. vol. 338, ed United States, 2012, pp. 1408-11.

Designing Novel

Protein 8

Riboswitches

Riboswitches Attached to

Transcription Factors:

Tunable Output

Levels

Variable Input

A. Serganov and E. Nudler, "A Decade of Riboswitches," Cell, vol. 152, pp. 17-24, 1/17/ 2013.

+ inducer

Protein

Design Criteria for

IdealTranscription Factors Typical

Transcription

Factor

RiboTALes

Sequence Specific Easily Engineerable

Target Specificity

Variable Input Usable in any Chassis

Building Our Testing

Constructs

Building Our Testing

Constructs

Testing Construct

pTET TBS

Testing Construct

pTET TBS

+ arabinose

Riboswitch

Testing Construct

pTET TBS

+ arabinose

Riboswitch

+ theophylline

Testing Construct

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

Experiment 1:

• Subject RiboTALe to a theophylline gradient

Riboswitch

+ theophylline

Translation is modulated

by theophylline

Experiment 2:

• Compare effect of TALe binding affinities on

system response

pTET TBS2

pTET TBS

TALe Kd = X

TALe Kd = Y

TAL repressor binding

affinities provide tunability

Experiment 3:

• Compare effect of riboswitch leakiness on

system response

Riboswitch

Riboswitch leakiness

modulates activity

Experiment 4:

• Full characterization of RiboTALe device

pTET TBS

+ arabinose

TALe Riboswitch

+ theophylline

3D RiboTALe Data

Characterization

25 With lots of data, what is the best way to share it?

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?

Scaling to All Parts

iGEM 2013UC Davis Human Outreach

Initial Population

Bielefeld-Germany

ZJU-China

Stanford-Brown

A Walkthrough: Uploading

A Walkthrough: Searching

A Walkthrough: Results

• After Downloading

• Able to Visualize

Output from Data Depot

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

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

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

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.

Thank You

UC Davis 20132013.igem.org/files/presentation/UC_Davis.pdf · iGEM 2013 UC Davis RiboTALs TALEs As a Transcription Factor: Sequence Specific Easily Engineerable Target Specificity

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