Bottom-up approaches in synthetic biology and biomaterials for tissue engineering applications

Abstract

Synthetic biologists use engineering principles to design and construct genetic circuits for programming cells with novel functions. A bottom-up approach is commonly used to design and construct genetic circuits by piecing together functional modules that are capable of reprogramming cells with novel behavior. While genetic circuits control cell operations through the tight regulation of gene expression, a diverse array of environmental factors within the extracellular space also has a significant impact on cell behavior. This extracellular space offers an addition route for synthetic biologists to apply their engineering principles to program cell-responsive modules within the extracellular space using biomaterials. In this review, we discuss how taking a bottom-up approach to build genetic circuits using DNA modules can be applied to biomaterials for controlling cell behavior from the extracellular milieu. We suggest that, by collectively controlling intrinsic and extrinsic signals in synthetic biology and biomaterials, tissue engineering outcomes can be improved.

Figure 1. Bottom-up approaches for controlling cell behavior

Synthetic biologists use interchangeable and well-characterized genetic parts, or modules, (listed in blue boxes) of DNA to build complex genetic circuits to program cells with robust and tunable gene expression for obtaining desired cell behavior. Synthetic biologists use a variety of genetic tools to target: (1) the nucleus, (2) the cytoplasm, and (3) the cell membrane to control gene expression that ultimately control cell behavior. The extracellular space (4) is another area for synthetic biologists to consider using a bottom-up approach to construct a cell-instructive microenvironment using well-characterized extracellular matrix (ECM) protein domains/modules (listed in yellow box). Together, controlling the intrinsic and extrinsic signals that cells are exposed to will likely improve our ability to guide the growth and behavior of cells.

Figure 2. Controlling matrix stiffness with peptide crosslinking modules

The stiffness of ECM scaffolds can be controlled by using modules that contain repeat motifs of either a “P” (proline rich – black triangles) or “C” (WW – red broken boxes). These modules can be mixed to control the stiffness of the matrix. A. When P and C domains are mixed, they form a soft hydrogel. B. When Nano HA modules are added to the P and C gels, the matrix stiffens. C. The addition of P-Decorated Nano HA modules enables an even stiffer matrix to be formed.

Figure 3. Coiled-coils

The primary structure of coiled coils can be modified to provide an unlimited number of these domains/modules to be used for structurally programming the ECM. A. Mixing PEG backbones (grey) conjugated with different coiled-coiled units (green, blue, yellow, red, and grey coils), the storage and loss moduli can be custom made to achieve desired ECM. B. The dynamic behavior of hydrogels linked via coiled-coil domains can be controlled by programming coiled-coil primary amino acid structures. C. A coiled-coil polymer system is capable of dissociation and re-association in response to the introduction of other coiled-coil domains. These networks can be disrupted through the introduction of a non-PEGylated coiled-coil domain with a higher affinity for one of the PEG-coil domains (boxed coiled-coils), thereby displacing the other PEG-coil binding partner from participating in crosslinking. D. When basic leucine zipper coils (Z_R) are fused to ELP (thick blue line) and mCherry (red sunshine) and mixed together, coacervates are formed to obtain slow, controlled release of mCherry-Z_E. E. The combination of collagen binding domains (red broken squares) and coiled coils provides a means to decorate the ECM with growth factors.

Figure 4. Spy Systems

Elastin like polypeptides ELPs (blue thick lines) harboring two SpyCatcher domains (red thick domain), an RGD region (green), and an MMP-cleavable sequence (purple) can be mixed with A. ELPs containing SpyTags (thinner red line) either flanking the ELP or placed internally, or B. with ELPs containing two terminal SpyTags along with LIF (yellow), a cytokine that aides in maintaining ES cell pluripotency. C. The SpyCatcher/SpyTag can be used to assemble functionalized peptides. D. SdyCatcher/SdyTag enables 3D architectures can be formed when cyclic SpyCatcher/SpyTag and cyclic SdyCatcher/SdyTag modules are mixed together.

Figure 5. Interfacing synthetic biology and biomaterials

A. LTRi_EGFP genetic circuit in the absence of inducer in is the off state and does not express Enhanced Green Fluorescence Protein (EGFP). B. When inducer is added, this flips the genetic switch on and EGFP is expressed. C. Genetically interactive biomaterials have been modified with genetic inducer molecules (blue circles) attached to the scaffold via photolabile bonds (orange lines). In the absence of 302nm of light, the inducer molecules remain attached to the biomaterial and do not activate EGFP expression. Upon exposure to 302nm of light, the photolabile bond breaks, releasing the inducer molecules, which activate EGFP expression. D. Cells harboring LTRi_EGFP were encapsulated into biomaterials and implanted into mice showed EGFP expression only when the genetic inducer was added to the drinking water.