Phenotypically complex living materials containing engineered cyanobacteria

Abstract

The field of engineered living materials lies at the intersection of materials science and synthetic biology with the aim of developing materials that can sense and respond to the environment. In this study, we use 3D printing to fabricate a cyanobacterial biocomposite material capable of producing multiple functional outputs in response to an external chemical stimulus and demonstrate the advantages of utilizing additive manufacturing techniques in controlling the shape of the fabricated photosynthetic material. As an initial proof-of-concept, a synthetic riboswitch is used to regulate the expression of a yellow fluorescent protein reporter in Synechococcus elongatus PCC 7942 within a hydrogel matrix. Subsequently, a strain of S. elongatus is engineered to produce an oxidative laccase enzyme; when printed within a hydrogel matrix the responsive biomaterial can decolorize a common textile dye pollutant, indigo carmine, potentially serving as a tool in environmental bioremediation. Finally, cells are engineered for inducible cell death to eliminate their presence once their activity is no longer required, which is an important function for biocontainment and minimizing environmental impact. By integrating genetically engineered stimuli-responsive cyanobacteria in volumetric 3D-printed designs, we demonstrate programmable photosynthetic biocomposite materials capable of producing functional outputs including, but not limited to, bioremediation.

3D Bioprinting of Microbial-based Living Materials for Advanced Energy and Environmental Applications

“…Natural polymers employed in 3D bioprinting are typically sourced from animals, plants, and algal microorganisms. On the basis of their chemical composition, they can be divided into polysaccharides (such as alginate, ,,,,, agarose, chitosan, hyaluronic acid (HA), , cellulose, methylcellulose, pectin, carrageenan, xanthan, gellan gum, cyclodextrin, , dextran, , etc.) and proteins (such as collagen, gelatin, , fibrin, silk fibroin, − etc.).…”

Engineered Living Structures with Shape-Morphing Capability Enabled by 4D Printing with Functional Bacteria

“…15,18,19 For instance, cellulose-producing bacteria were encapsulated in a hyaluronic acid gel or silicone-based granular gel, and the 3D-printed structures had the capability to self-regenerate cellulose fiber networks and demonstrate self-healing. 17,20 Cyanobacteria were incorporated in a gel matrix and 3D-printed into ELMs with well-defined shapes; 21 those ELMs depend on only light energy and CO 2 as a carbon source for growth and can be used in bioremediation. Genetically engineered E. coli has also been incorporated in alginate and 3D-printed onto calcium-containing substrates, 22 T h i s c o n t e n t i s and patterned biofilm formation with curli fiber formation was demonstrated.…”

“…In order to create ELMs with well-defined and customizable shapes, geometry, and functions, recently, three-dimensional (3D) printing has been increasingly used for generating these materials. ,, For instance, cellulose-producing bacteria were encapsulated in a hyaluronic acid gel or silicone-based granular gel, and the 3D-printed structures had the capability to self-regenerate cellulose fiber networks and demonstrate self-healing. , Cyanobacteria were incorporated in a gel matrix and 3D-printed into ELMs with well-defined shapes; those ELMs depend on only light energy and CO 2 as a carbon source for growth and can be used in bioremediation. Genetically engineered E.…”

Growth, Distribution, and Photosynthesis of Chlamydomonas Reinhardtii in 3D Hydrogels

“…[5,6] These observations suggest that simply increasing the total volume of ELMs would not necessarily lead to higher functionality/productivity. Photosynthetic microorganisms can be integrated into polymeric matrices to form ELMs with plant-like qualities, [7][8][9][10][11][12][13][14][15][16][17] allowing them to photosynthesize and thus to provide a localized O 2 source [12,15] and/or CO 2 sink. The high tolerance of many microalgae to abiotic stress factors such as temperature, pH, or osmotic stress broaden the spectrum of potential applications for photosynthetic ELMs.…”

“…The high tolerance of many microalgae to abiotic stress factors such as temperature, pH, or osmotic stress broaden the spectrum of potential applications for photosynthetic ELMs. Based on these properties, microalgal ELMs show great promise for generating bioelectricity, [14] oxygenating mammalian tissues in biomedical applications, [15] improving air and water quality, [7,9,16] and granting photoresponsive/photoadaptive functions to materials. The current research on fabrication of microalgal ELMs has so far focused on formulation of hydrogel, [10] viability of embedded cells, [8,10,15] and O 2 production capacity in dependence of time.…”