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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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Page 1: 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

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Page 2: 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

iGEM 2013 UC Davis RiboTALs

Synthetic Biology

INPUT OUTPUT

Controlled by Transcription Factors

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Page 3: 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

iGEM 2013 UC Davis RiboTALs

Need for Input Diversity

• Controlled by Transcription Factors

5 5n

INPUTS OUTPUTS

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Page 4: 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

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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Page 5: 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

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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Page 6: 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

iGEM 2013 UC Davis RiboTALs

Designing Novel

Transcription Factors

DNA

RNA

Protein 6

Page 7: 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

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.

7

Page 8: 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

iGEM 2013 UC Davis RiboTALs

Designing Novel

Transcription Factors

DNA

RNA

Protein 8

Page 9: 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

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.

9

+ inducer

Protein

Page 10: 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

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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Page 11: 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

iGEM 2013 UC Davis RiboTALs

Building Our Testing

Constructs

Page 12: 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

iGEM 2013 UC Davis RiboTALs

Building Our Testing

Constructs

Page 13: 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

iGEM 2013 UC Davis RiboTALs

Testing Construct

TALe

pBAD

GFP

pTET TBS

+ aTc

Page 14: 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

iGEM 2013 UC Davis RiboTALs

Testing Construct

TALe

pBAD

GFP

pTET TBS

+ aTc

+ arabinose

TALe

Riboswitch

Page 15: 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

iGEM 2013 UC Davis RiboTALs

Testing Construct

TALe

pBAD

GFP

pTET TBS

+ aTc

+ arabinose

TALe

Riboswitch

+ theophylline

TALe

TALe

Page 16: 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

iGEM 2013 UC Davis RiboTALs

Testing Construct

Page 17: 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

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

Page 18: 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

iGEM 2013 UC Davis RiboTALs

Experiment 1:

• Subject RiboTALe to a theophylline gradient

TALe

Riboswitch

+ theophylline

TALe

Page 19: 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

iGEM 2013 UC Davis RiboTALs

Translation is modulated

by theophylline

Page 20: 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

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

Page 21: 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

iGEM 2013 UC Davis RiboTALs

TAL repressor binding

affinities provide tunability

Page 22: 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

iGEM 2013 UC Davis RiboTALs

Experiment 3:

• Compare effect of riboswitch leakiness on

system response

TALe

Riboswitch

TALe

Riboswitch

Page 23: 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

iGEM 2013 UC Davis RiboTALs

Riboswitch leakiness

modulates activity

Page 24: 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

iGEM 2013 UC Davis RiboTALs

Experiment 4:

• Full characterization of RiboTALe device

TALe

pBAD

GFP

pTET TBS

+ aTc

+ arabinose

TALe Riboswitch

+ theophylline

TALe

Page 25: 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

iGEM 2013 UC Davis RiboTALs

3D RiboTALe Data

Characterization

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

Page 26: 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

iGEM 2013 UC Davis RiboTALs

Modeling

• Mathematical support

for device functionality.

• Can be developed into

a predictive tool for

RiboTALe design

Page 27: 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

iGEM 2013 UC Davis Human Outreach

Raw Data for RiboTALs

• Complex system with

multiple inducers

• How can we best share?

Page 28: 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

iGEM 2013 UC Davis Human Outreach

Page 29: 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

iGEM 2013 UC Davis RiboTALs

Scaling to All Parts

Page 30: 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

iGEM 2013UC Davis Human Outreach

Initial Population

Bielefeld-Germany

ZJU-China

Stanford-Brown

UCSF

Page 31: 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

iGEM 2013 UC Davis Human Outreach

A Walkthrough: Uploading

Page 32: 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

iGEM 2013 UC Davis Human Outreach

A Walkthrough: Searching

Page 33: 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

iGEM 2013 UC Davis Human Outreach

A Walkthrough: Results

Page 34: 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

iGEM 2013 UC Davis Human Outreach

• After Downloading

Data:

• Able to Visualize

Output from Data Depot

Page 35: 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

iGEM 2013 UC Davis Human Outreach

Page 36: 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

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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Page 37: 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

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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Page 38: 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

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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Page 39: 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

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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Page 40: 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

iGEM 2013 UC Davis RiboTALs

Thank You

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