Michigan Synthetic Biology Team

2013

Introduction Mike Ferguson

What is a Transcriptor? What is its utility?

Sources: Jerome Bonnet et al. Amplifying Genetic Logic Gates. Science 3 May 2013, Vol. 340 no. 6132 pp. 599-603. Jerome Bonnet, Pakpoom Subsoontorn, and Drew Endy. Rewritable digital data storage in live cells via engineered control of recombination directionality. PNAS. 2012 Apr 6.

Int/Ex Transcriptor Logic Gates

●  Regulates flow of RNA polymerase across strand of DNA

●  Used in logic gates

●  Tightly controlled switch or as a storage device

The Problem with Current Transcriptors

•  Recombination Directionality Factors

•  Not completely unidirectional

Source: Jerome Bonnet, Pakpoom Subsoontorn, and Drew Endy. Rewritable digital data storage in live cells via engineered control of recombination directionality. PNAS. 2012 Apr 6.

The Solution: Utilizing the Fim Switch

Source: M. P. McCusker, E. C. Turner and C. J. Dorman. DNA sequence heterogeneity in Fim tyrosine-integrase recombinase-binding elements and functional motif asymmetries determine the directionality of the fim genetic switch in Escherichia coli K-12. Molecular Microbiology, 67, 171–187.

Caveat: DNA bending proteins and other Regulators

Source: I. C. Blomfield, D. H. Kulasekara and B. I. Eisenstein. Integration host factor stimulates both FimB- and FimE-mediated site-specific DNA inversion that controls phase variation of type 1 fimbriae expression in Escherichia coli. Molecular Microbiology (1997) 23(4), 705–717.

Source: 2012 Michigan iGEM

Source: Schwan WR. Regulation of fim genes in uropathogenic Escherichia coli. World J Clin Infect Dis 2011; 1(1): 17-25.

Wet Lab: Design Drew Dunham

Switch Design and Sequence Determination

●  Conserving the LRP and IHF binding sites ●  Deletion of the most of the native fimA promoter

○  Allows for a switch with customizable promoter function insertion

Source: D. L. Gally, J. Leathart and I. C. Blomfield. Interaction of FimB and FimE with the fim switch that controls the phase variation of type 1 fimbriae in Escherichia coli K-12. Molecular Microbiology (1996) 21(4), 725–738.

Assaying the Transistor: Chromophore Reporter

●  Utilization of multiple chromophore reporters ○  GFP

■  Allows for easy quantification due to highly characterized nature

■  Requires UV excitement to visualize ○  AmilCP

■  Easily visualized by the naked eye ■  Not a fluorescent protein

●  Utilizing repeated restriction enzyme cut sites to identify the state of the switch

Assaying the Transistor: Digest

B A

A

B

Inducing Recombinase Activity

Wet Lab: Results Mike Ferguson

The Fim Transcriptor is Capable of Changing States Completely and Unidirectionally

•  Constitutively expressed fimE and hbiF flip the engineered switch completely

•  Faint bands may be due to too high copy plasmid number or the recombinase generator itself in the case of the ON lane

An Inducible Fim Transcriptor System Changes States and Produces Protein Output

•  Uninduced colonies show a mixed state

•  The switch can still produce protein

Determination of Inducer Concentrations Necessary to Flip the Fim Transcriptor Completely

•  HSL flips the switch ON, producing amilCP

•  aTc flips the switch OFF, producing no protein

•  The color of the bacteria is not enough to determine how efficient the state change was.

•  7uM aTc flips the switch completely •  The concentration of HSL still needs to be determined.

An Inducible Recombinase Generator is Capable of Cycling the State of the Fim Transcriptor

•  The fim transcriptor can cycle states

•  The grayish color in 4B may be due to incomplete protein degradation

•  Given more time, flipping back to the OFF state does not show any residual amilCP

Results Discussion ●  It works! ●  Need to do the digest assay on the cultures

with higher HSL concentrations ●  High copy plasmid may result in not all of

the switch being flipped

Future Directions Raoul Martin

Future Directions •  Chromosome integration

o  Eliminate the use of high copy plasmid.

o  Integrate the switch in the E. coli chromosome

o  The cell will only be able to be in an “ON” or “OFF” state.

Reasoning: Eliminate the chances of expressing “ON” and “OFF” switch states simultaneously.

Future Directions ●  In vitro assay:

○  Purify both HbiF and FimE

○  Add one of the recombinases to a solution containing the switch plasmid.

○  PCR purification ○  PCR amplification

Reasoning: This will enable us to check if HbiF and FimE are the only two proteins required for the switch to function.

Dry Lab: Modeling Joshua Abramson

Switch Modeling

Models

●  Expression model

●  Inducible Hbif model

●  Switch model

Modeling Approach

●  Mass action differential equation systems

●  Analytical vs. numerical modeling

ERSESCO Algorithm

7. Optimization

4. Equilibration

6. Calibration

5. Stabilization

3. Solution

2. Reduction

1. Equation

Switch Model

• Two irreversible catalyses • Pseudo-steady state enzymes • Enzyme cooperativity

Switch Model

• Equation:

• Reduction:

• Solution:

• Equilibration:

• Stabilization:

Switch Model

• Calibration:

• Optimization:

Other Models

Human Practice Corey Howe

●  University of Toledo, OH ●  Ocean and Ivor Huang ●  Talked modeling with Team Toulouse

WISE ●  Collaboration with U of M Program. ●  Women In Science and Engineering. ●  Experiments

- Strawberry DNA extraction - Saliva DNA extraction and necklace - Gel Electrophoresis

●  Presentations and Quizzes. ●  Celebrity Crime Mystery

Outside Help

●  Educational Platformer Video Game ●  Mobile App and Computer game ●  Plasmid Paul Character ●  Broad Audience Range ●  Original Soundtrack ●  Currently in Beta Version ●  Can be played on our Wiki Page

Adventures of Plasmid Paul

Adventures of Plasmid Paul

Ligation Level

Freezer Level

Zombie News Freezer Level Incubator Level

Mini-Prep Level Digestion Level Ligation Level

PCR Level Gel Level Boss Level

While 32 people played the game…

●  83% of players enjoyed the time they spent. ●  32% of players learned what a protein is.

●  22% of players learned what a plasmid is. ●  31% of players learned what synthetic biology is.

●  66% of players interest in synthetic biology was sparked.

●  Before the game, 3% of the players thought synthetic biology was more harmful than helpful to society, while after the game none of the players thought it was more harmful.

●  10% changed their thoughts to think that synthetic biology is safe.

●  83% of players learned something from the game.

Results

Conclusion Jonah Sementkowski

• Created a working fim transcriptor and demonstrated greater than 95% efficiency on a high copy plasmid

• Developed an effective educational video game

• Defined a new procedure for analysis of mathematical systems

• Made the fim transcriptor easily customizable

• Submitted a complete, inducible, fim transcriptor system

• Submitted characterization data for numerous new and improved registry parts

Accomplishments

Inducible fimE and hbiF recombinase generator (tet and lux)

fim switch inverted repeat left (IRL) natural

fim switch inverted repeat right (IRR) natural

Submitted Parts

J23100 fim switch B0034 amilCP ON orientation

J23100 fim switch B0034 GFP ON orientation

J23100 fim switch ON orientation

Natural fim switch OFF orientation

Natural fim switch B0034 GFP OFF orientation

Submitted Parts

Thank you! Questions?