Team:William and Mary/Description
Project Description
Abstract
Our iGEM project aims to create a toolkit for the precise, controlled design of living biomaterials. Of the many forms of living materials, we have chosen bacterial biofilms due to their ubiquity and outstanding bioengineering potential. Biofilm formation, although frequently associated with deleterious effects, also equips bacteria with emergent properties such as increased resilience, complex signaling, self-repair, and division of labor. Our project harnesses these properties to repurpose biofilms as robust, spatially controlled, patterned, and responsive biomaterials. For robustness, we engineered a library of biofilm-strengthening adhesins, and investigated naturally biofilm-forming bacterial species. To adhere biofilms with precise spatiotemporal control, we incorporated both optogenetic and chemical induction methods. To pattern biomaterials once placed, we utilized the distance-dependent diffusion of quorum signaling molecules and investigated Turing patterns that are informed by mathematical modeling. Our biomaterials have immediate applications in wound healing, water and waste treatment, and the creation of next-generation biosensors.
Inspiration
Jason Kelly, CEO of bioengineering company Ginkgo Bioworks, spoke at the iGEM Jamboree last year. He presented the audience with an image of his office desk and asked, of all of his belongings, which was the most sophisticated. Of course, it was the houseplant, over advanced technology such as his computer. Unlike even the most complex computers, the plant can respond to its environment, grow, and regenerate. Living Biomaterials, or engineered materials composed of living cells, can accomplish what standard materials cannot. Inspired by his speech, we began consulting the literature on biomaterials and programmed biofilms. Eventually, we found the paper “Biofilm lithography enables high-resolution cell patterning via optogenetic adhesin expression” by Stanford bioengineers Jin & Riedel-Kruse (2018). Inspired by their ability to place biofilms with high resolution and control, we began designing programmed biofilms. We used their plasmid, pDawn-AG43, as a starting point for our experience with engineered biofilms. Many of our toolkit components arose as a means of building upon pDawn-AG43 or strengthening it.
Motivation
Why living biomaterials?
In their review “Engineered Living Materials: Prospects and Challenges for Using Biological Systems to Direct the Assembly of Smart Materials,” Nguyen et al. define engineered living materials as “engineered materials composed of living cells that form or assemble the material itself, or modulate the functional performance of the material in some manner” (2018). Engineered living materials compose one class of biomaterials, which are natural or synthetic materials that “interface with biological systems” to improve function or replace components (Tibbitt & Langer, 2017). We have chosen to engineer living biomaterials because they exceed both standard nonliving materials and nonliving biomaterials. After tedious, manual assembly, nonliving materials proceed to wither and break, demanding repair. In contrast, living biomaterials can self-assemble, regenerate, and repair (Chen et al., 2015). Furthermore, living biomaterials and “smart” nonliving biomaterials can perceive and respond to their environment, adapting and behaving appropriately (Kowalski et al., 2018). When combined with standard nonliving materials, living biomaterials can perform complex, specialized functions, such as conducting electricity or degrading toxic metals.
Recent studies have employed the unique qualities of biomaterials in various fields. Perceptive and responsive, biomaterials adapt to their environments, and operate well in applications that demand precise induction. For example, medical researchers have created biomaterials that only release drugs or cancer-marking tags when triggered by certain physical parameters (Tibbitt and Langer, 2017). The applications of environmentally-perceptive biomaterials span far beyond medicine, however. By placing a synthetic curli operon under the control of a mercury-sensitive promoter, biofilm researcher Neel S. Joshi designed a mercury-sequestering biofilm to aid in bioremediation (Tay et al., 2017).
Along with their ability to perceive and respond to their environment, living biomaterials’ ability to self-repair and regenerate holds promise for countless applications. Their regenerative ability eliminates the costly, labor-intensive maintenance required by standard nonliving materials. For instance, medical researchers have designed “cell-laden hydrogels” that successfully integrate with tissue and deliver glucose to diabetes patients for up to six months (Tibbitt and Langer, 2017). Meanwhile, civil engineers have investigated living biomaterials as the key to self-repairing concrete (Patel, 2015).
Why pattern living biomaterials?
Patterning is ubiquitous in nature. Though famous examples include the Hox genes that allow for stunningly-complex eukaryotic life as we know it, even relatively simple, single-celled organisms exhibit spatiotemporal heterogeneity in gene expression, structure, and function. Bacteria, for example, rely on gradients of molecules to “know” exactly where to form a septum and divide the cell in half. As patterning is inherent to living organisms, we must pattern living biomaterials in order to exert complete control over their gene expression and related function. Not only does patterning control biomaterials, but also it expands upon their ability to develop sophisticated structures and accomplish complex tasks. An unpatterned material expressing the same genes in the same location for all time can only accomplish so much.
Current research has ventured into the realm of patterning for improved applications. One study found that a multi-layered material scaffold increased desirable cell differentiation in human tissue, suggesting that the ideal scaffold should combine “several biomaterials, each contributing specific property to design a superior material” (Kowalski et al., 2018). Since patterning enables differential gene expression within a biofilm, it can establish division of labor and mimic natural systems when applied to tissue engineering. Researchers have accomplished this type of patterning using bioprinting, which can create scaffolds with varying components, porosity, and geometry. Other methods of patterning could also be applied to precisely-controlled microbial consortia, which has applications ranging from chemical synthesis to biological computers used in the diagnosis of diseases and creation of “smart drugs” (Kahan et al., 2008). Microbial consortia can be patterned to carry out logic gate functions, in which they produce different inputs that lead to an output (Anderson et al. 2007). Researchers Bonnet et al. utilized transcriptor-based logic gates to create a biological transistor, a component of biological computers (2013).
The state of programmed biofilms
Existing research describes patterned, regenerative, and perceptive biomaterials, but synthetic biology still lacks a toolkit to utilize biofilms as these living biomaterials. Though the majority of bacterial and archaeal cell exist within biofilms, biomedical bias has generated historically negative perspectives towards biofilms, which many studies aim to prevent and eradicate. Studies that do utilize and design biofilms provide specific, targeted techniques, but stop far short of a complete toolkit.
One extremely well-characterized technique available in the literature is curli manipulation. Biofilm expert Dr. Neel S. Joshi has manipulated curli fiber production extensively, fusing proteins to curli subunit csgA to confer novel qualities (such as protein sequestration and substrate-specific adhesion) to biofilms. As mentioned previously, he has also placed curli under the control of a mercury-sensitive system to employ biofilms for bioremediation (Tay et al., 2017). TU-Delft researcher Dr. Anne Meyer has also utilized curli, which she incorporates into printed biofilms within an alginate matrix (Balasubramanian et al., 2019)
Our Toolkit
To expand upon the existing literature and constitute a biomaterials toolkit, we have explored more methods of biofilm induction and adhesion. As well, to fully utilize biofilms as biomaterials, we have investigated methods of patterning for tighter spatial, temporal, and behavioral control, as well as more sophisticated functions. One such method of patterning we found in the literature is “Biofilm Lithography,” a method of light-induced biofilm adhesion coined by Stanford researchers Jin and Reidel-Kruse (2018). Though this method accomplishes high-resolution spatial patterning, it relies on AG43, a weak adhesin that often results in premature biofilm flaking and disintegration.
To build upon Biofilm Lithography as well as Dr. Meyer and Dr. Joshi’s work, we explored a library of biofilm forming and strengthening adhesin proteins. This library includes not only curli fibers, which are native to E. coli, but also adhesins native to Pseudomonas and Staphylococcus. We placed such fibers into 3G-Assembly compatible format and assembled optogenetic, chemically-induced, and constitutive circuits. We engineered multiple methods of induction in order to overcome the limitations of previous techniques. For example, Biofilm Lithography requires 16 hours of overnight growth and light access to the substrate. Though faster, Dr. Meyer’s bioprinter requires physical access to the substrate, and places cells under physical stress when extruding them from the nozzle. Finally, expression of our adhesins was quantified using Congo Red staining and further visualized via microscopy. We realize, however, that biofilm potential is limited for non-native biofilm forming bacteria such as E. coli. After consulting microbiology expert Dr. Mark Forsyth, we investigated natural biofilm former Mycobacterium smegmatis. We placed various mycobacterium parts in 3G Assembly-compatible format and began characterization, so that this non-model organism may be better utilized as a chassis.
To pattern our biofilms once formed, we engineered quorum sensing to create a gradient of signal molecules and thus a gradient of responses, resulting in distance-dependent patterning. As mentioned previously, patterning controls programmed biofilms while expanding upon their potential structures and functions. Patterning opens applications such as tissue engineering and microbial consortia, which require division of labor in the correct proportions. Patterning biofilms for biomaterials could also allow for new and advanced technology, such as complex biological electrical circuits. Studies have accomplished electricity-conducting curli nanofibres; if researchers carefully patterned and constructed these, they could create a completely biological electrical circuit (Courchesne et al., 2018).
Specifically, applications for our toolkit include a patch for the treatment of chronic wounds. While smart materials exist to measure wound pH and temperature, programmed biofilms could detect and outcompete dangerous microbes, provide readable outputs on wound healing progress, and accelerate the healing process by synthesizing and delivering beneficial compounds. Here, patterning would be essential. The spatially heterogeneous nature of wounds requires two-dimensional patterning, while patterning on a three-dimensional scale would allow biofilm layers to play different but equally integral roles, similar to layers of natural human skin. Layers closest to the wound could interact with cytokines and other inflammation-related substances to accelerate healing, while layers exposed to the outer environment could function in a protective capacity.
However, this is one potential direction. Since our 2019 project encompasses a true biomaterials toolkit, the possible future applications are limitless.