We thank Prof. Ron Weiss for his advice and financial support, Jonathan Babb, Ph.D. and Deepak Mishra for their instruction and insight, the Elowitz group at Caltech for cell lines and DNA, and our corporate sponsors EMD-Millipore, Genewiz, Geneious, Ginkgo Bioworks, Addgene, EmbiTec, BBN Technologies, EBICS and New England Biolabs for their support.

Our system is a novel approach to tissue engineering, combining signaling,

internal logical design, and adhesion.

Current approaches of tissue engineering are largely characterized by external design, such as the precise printing of cells or selective induction by light.

vs.

Our approach differs in that it uses intercellular communication and modular internal processing to achieve self-assembly and patterning of cells.

The Notch-Delta signaling pathway is inherently interesting for its cis-inhibition and trans-activation characteristics, and is a highly-conserved pathway involved in the development of many organisms. Cis-inhibition is important for groups of cells to achieve different fates and form complex structures.

Cadherins are responsible for cell-to-cell adhesion and play a crucial role in tissue and organ formation.

Figure 3. Modeling the Patterning Process. Simulation using our framework, mCell, comparing naturally adhesive and non-adhesive cells containing our N-cadherin circuit. The last image shows developed borders and Delta expression along the boundaries.

Figure 1. Circuit. This circuit illustrates our three main components: signaling, internal logic processing, and adhesion.

Hef1a Gal4-VP16 UAS LacI Hef1a-LacOid rtTA3

IPTG Gal4-VP16 LacI

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Two-Input chemical AND Gate

HIGH-LEVEL MOTIVATION

DESIGN

SIMULATIONS, CHARACTERIZATIONS, & RESULTS

REFERENCES

SUMMARY & ACHIEVEMENTS

ACKNOWLEDGEMENTS

1. Engler C, Kandzia R, Marillonnet S, 2008 A One Pot, One Step, Precision Cloning Method with High Throughput Capability. PLoS ONE 3(11):e3647. doi:10.1371/journal.pone.0003647

2. Sprinzak D, Lakhanpal A, LeBon L, Garcia-Ojalvo J, Elowitz MB, 2011 Mutual Inactivation of Notch Receptors and Ligands Facilitates Developmental Patterning. PLoS Comput Biol 7(6): e1002069. doi:10.1371/journal.pcbi.1002069

Figure 5. Circuit Design of two-input AND Gate with doxycycline and IPTG.

MIT IGEM

METHODS

DNA Construction Techniques:

Golden Gate Assembly (ligation-based)

Gibson Assembly (chewback and anneal based)

Gateway Assembly (recombinase based)

Transfection Techniques into Mammalian Cells:

Transient with Lipofectamine 2000 (Invitrogen)

Nucleofection (Lonza)

Before the construction of our circuit, we modeled pattern formations. Our simulation in Figure 2 shows that linking the Notch-Delta system and cadherins to our internal logic processing system can lead to the formation of interesting tissue patterns over time. Our results in Figure 3 demonstrate that this combination prompts pattern formation in mammalian cells.

HUMAN PRACTICES

Figure 4. Activation of Notch and Delta. We tested Notch-Delta signaling by mixing sender and receiver cells and adding doxycycline to begin Delta-mCherry expression. Delta activates Notch and citrine expression. The addition of doxycycline significantly increased red fluorescence from senders and yellow fluorescence in receiver cells. Expression in stably integrated Elowitz cells was used as a positive control (row 3). Receiver cells with blank sender cells were used as a negative control (row 2).

This is one of our internal processing modules: a two-input chemical AND gate. We combined three transcription factor systems into a two chemical AND gate with IPTG and doxycycline inputs. IPTG uninhibits rtTA3, which then requires doxycycline to activate the TRE promoter. Therefore, both IPTG and doxycycline are needed to achieve expression of the final Delta-mCherry reporter.

Figure 6. Two-input IPTG and doxycycline AND gate. The results confirm that in the absence of doxycycline, rtTA3 cannot activate the TRE promoter despite an increase in IPTG concentration,

Our 2-Week SynBio Class:

Taught basic laboratory techniques. Emphasized the diversity of SynBio. Created awareness of problems and possibilities that exist

within SynBio today. Encouraged excitement about SynBio in members of the

Greater Boston community who were unfamiliar with biological engineering.

Collaboration with BU-Wellesley Team: Assisted them in creating an ambient auxiliary system for

the laboratory setting. Helped them bridge the designer and end-user gap. Provided them with information for the HCI (Human-

Computer Interaction) aspects of their project.

The data demonstrates the desired 2-input AND gate behavior. We observe maximum Delta-mCherry expression at the highest levels of both doxycycline and IPTG. We also see activation at high doxycycline levels even in the absence of IPTG, suggesting incomplete lacI repression. The data was collected using flow cytometry, which measured red fluorescence from Delta-mCherry expression.

Multi-Input Logic Characterization

Simulations & Patterning

N-Cadherin Mediated Cell Adhesion

Characterization of six new mammalian transactivators, four new mammalian repressors, and four new reporters optimized for mammalian expression.

Engineering of the first mammalian communication system, based on Notch and Delta proteins.

Adhesion of mammalian cells due to expression of the cadherin adhesion protein, unlocking the key to autonomous patterning.

Assembly of components for an orthogonal signaling pathway designed for synthetic juxtacrine signaling.

Modeling of multicellular behavior in response to synthetic circuits through the new mCell modeling platform.

Framework for future mammalian work through twenty seven new parts submitted to the Registry.

40X

CHO+ NCad- eGFP

GFP control

Cadherins are medically important calcium-dependent surface proteins that adhere to similar cadherins on other cells. The existence of different types of cadherins allows for the development of highly organized tissues, as the differential expression of different types of cadherins in various cells drives the development of complex three-dimensional structures.

We've incorporated cadherins into our characterized signaling work in order to achieve control of cadherin expression in mammalian cells. By integrating cadherins into our pattern-formation mechanism, we can now create self-adhesive structures, resulting in a new engineering platform for regenerative medicine and tissue engineering, and leading to programmed organogenesis.

Figure 7. Cadherin expression and adhesion. As shown in the microscopy images to the left (A and B), our N Cadherin-GFP fusion shows clear membrane localization. In addition, fluorescent cells adhere with intense GFP localization at cellular interfaces. Control cells transfected with GFP alone show diffuse cytoplasmic localization and no preference for sticking.

Team Members: Divya Arcot, Jenny Cheng, Jonathan Chien, Michelle Dion, Kenneth Hu, Tiffany Huang, Charles Hsu, Clara Park, Semon Rezchikov, Grant Robinson, Mariola Szenk, Tyler Wagner

CHO Sender HEK Receiver Combined 2ug/mL dox

Blank CHO HEK Receiver Combined 2ug/mL dox

CHO Receiver CHO Send+Recv CHO Send+Recv +Blank Dox=0 Dox=2ug/mL

Pattern with developed boundaries.

Figure 2. Gateway LR Reaction.

A B