Overview CharacterizaTIon of Sender CellsS Characterization of Receiver CellS

OPTICAL COUPLING OF BIOLOGICAL CIRCUITS

Converting Between Relative units and Intensity

Future Directions

References1. Amy Gutmann, “The Ethics of Synthetic Biology: Guiding Principles for Emerging Technologies,” Hastings Center Report 41, no. 4 (2011): 17-22.2. Andrianantoandro, E., Basu, S., Karig, D. K., &Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Molecular Systems Biology, 2, 2006.0028. http://doi.org/10.1038/msb41000733. Craney A, Hohenauer T, Xu Y, Navani NK, Li Y & Nodwell J (2007) A synthetic luxCDABE gene cluster optimized for expression in high-GC bacteria. Nucleic Acids Res 35: e46.

4. "Team:CU-Boulder/Project." Team:CU-Boulder/Project. N.p., n.d.Web. 18 Sept. 2015. Journal of Molecular Biology 416.4 (2012): 534-42.Web.5. "PDusk and PDawn - Light Controlled Fluorescent Protein Expression." The ODIN. N.p., n.d. Web. 18 Sept. 2015.6. Yagur-Kroll S, Belkin S. 2011. Upgrading bioluminescent bacterial bioreporter performance by splitting the lux operon. Anal. Bioanal. Chem. 400: 1071-1082

ACKNOWLEDGEMENTS

Sponsors

Undergraduate MembersNikita Rao, Jane Shmushkis,

Karol Szymula, Shriram Sundararaman

AdvisoRDr. Brian Chow

MentorS

Language of Lightthe

A biological analog for the optocouplerThe Dilemma

The Electrical Optocoupler

The Biological Optocoupler

Communication between cells is instrumental in coordinating population-level activity, but current mechanisms of cell-cell communication rely on chemical signal transduction.

Chemical signals are limited by di�usion and require cells to be growing in a common environment, but there are many cases where organisms cannot be reliably co-cultured or require physical seperation.

How can synthetic biologists overcome this design constraint?

RLU is a measurement of photon count unique to each individual instrument. It is desirable to correlate the RLU obtained from the plate reader to a universal unit of irradiance(uW/cm2), enabling comparisons of values across di�erent systems

Due to the di�erence in sensitivity ranges of the plate reader and the power meter, a luminometer was used to bridge the sensitivity gap between the two instruments. Two calibration steps (shown below) are therefore required to establish the relationship between RLU measured by the plate reader and irradiance

In electronics, an optocoupler is a circuit component that transfers electrical signals between two isolated circuits using light

Circuit A

Light-Emitting Diode

Phototransistor

Circuit B

Examples include isolating a microcontroller from a high-voltage input signal, and turning on a TV with a remote control.

A biological optocoupler would allow for cell-cell communication between isolated organisms and would overcome the design contraint imposed by di�usion

Sender Cell Receiver Cell

Steps for Building a Biological Optocoupler

Figure 1: A schematic diagram of a simple optocoupler consisting of an LED and a phototransistor. When Circuit A is “ON,” the LED emits light which triggers the phototransistor and turns Circuit B “ON”.

Figure 2: A schematic diagram of a biological optocoupler consisting of a Sender Cell and a Receiver Cell. When the Sender Cell is “ON,” the cell expresses a luciferase enzyme which emits light and triggers a Receiver Cell which has been engineered to be light-responsive.

Characterize the luminescence generated by di�erent strains of sender cells

Convert relative measurement of luminescence to absolute values of irradiance

Characterize illumination sensitivity of receiver strain and compare to obtainable irradiance from Sender cells

Establish optical coupling of sender and receiver cells and evaluate predictability of the system based on individual characterizaiton

C D EA B

LUX OPERON

Figure 3: The Lux Operon is used to enable sender cells to emit light through a chemiluminescence reaction. a) The Luxbrick (BBa_K325909, Cambridge iGEM 2010) allows inducible control of the entire Lux operon b) split lux system places the luxAB genes under inducible control while constitutively expressing luxCDE. c) The Luxbrick was transformed into a standard NEB10 strain of E. coli as well as an H-NS mutant, which has the H-NS protein knocked out that has been shown to repress expression of the lux operon. The split-lux system was transformed into strain SY104.

The Lux Operon

C

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

0 5 10 15

RLU

/OD

600

Time (h)NEB10 HNS SY104 NEB10 fit HNS fit SY104 fit

The Sender Strains Luminescence Output over Time

A B

Luciferase

Pinducible

Pinducible

C D E

Pon

Enzymes involved in co-factor synthesis

a)

b)

NEB10

HNS

SY1046

c)

Figure 4: Sender Cell Luminescence Time Course. NEB10 and HNS mutant exhibit high peak luminesence with rapid decay, while SY104 luminesnce is sustained over longer amount of time.

PLATEREADER

LUMINOMETER

LUMINOMETER POWERMETER

RELATIVE LUMINESCENCE UNIT (RLU)Measured by a luminometer

or plate reader

INTENSITY (uW/cm2)Measured by a powermeter

pDawn, a light-gated transcription regulator, is expressed in receiver cells. pDawn activitation under blue illumination is measured through RFP reporter expression.

Increase in RFP expression after 15h suggests successful communication between all sender-receiver pairings SY104 yielded the highest receiver activation, suggesting that sustained luminescence is preferred for activation of pDawn. This is consistent with the slow activation kinetics of pDawn shown in Ohlendorf et al. 2011

Alter promoter strength to increase yield of luminescence from sender

Tune the photoreceptor to increase sensitivity of receiver

Establish a light-based communication system that extends across di�erent species (i.e. bacteria and yeast)

Predictability of the systemPredicted RFP expression with SY104 strain agrees with experimental observation, while RFP expression with NEB10 and HNS are over-predicted.Our computation can predict the behavior of the system more accurately if the senders exhibit sustained luminescence.

Spencer Glantz, Pimkhuan Hannanta-anan, Michael Magaraci

Sustained Intensity of SY104

0.28 uW/cm2

Peak Intensity of NEB10

1.57 uW/cm2

Peak Intensity of HNS

1.83 uW/cm2

By calibrating the plate reader against the luminometer, and calibrating the luminometer against a powermeter, we were able to obtain approximate intensity values for our sender strains.

Dose-response curve of pDawn shows agreement with published pDawn activation in Ohlendorf et al. 2011

Based on sender characterization, SY104 senders are estimated to produce 0.28 uW/cm2 in illumination intensity. This is su�cient for pDawn activation in receiver.

To evaluate the light-based communication, we cultured senders and receivers (arrangement below), added inducers to sender cells, and exposed the receiver to receiver luminescing culture. RFP fluorescence, luminescence, and OD600 were measured at di�erent times post-induction.

Error bars represent standard deviation, n=3

Error bars represent standard deviation, n=3

Conversion of luminescence measured by Tecan plate reader to irradiance

Predicted Fold Change in RFP Expression in Response to Sender Luminescence

Analysis of Predicted Fold Change in RFP Expressions vs. Experimental Communication Results

The Penn 2015 iGEM team would like to thank our mentor Dr. Brian Chow and our advisors Spencer Glantz, Pimkhuan Hannanta-anan, and Michael Magaraci for their continued guidance and support through the entirety of our iGEM experience. We would also like to thank GenScript and Integrated DNA Technologies for providing us with essential resources to complete our project. We also would like to give a

special thanks to Dr. Mark Goulian and The Hebrew University of Jerusalem for contributing to our project by donating plasmid vectors that we needed. Finally, we want to thank the Stephenson Bioengineering Undergraduate Lab at the University of Pennsylvania for providing with lab space and materials to execute our project.

RFP

pDawn

luminescent sender

surrounding cuvette

cuvette with light-sensitive

receiver

50 mL

centrifuge tube