An official website of the United States government
Abstract Herein, a method that uses direct‐ink‐write printing to fabricate engineering living materials (ELMs) that respond by undergoing a programmed shape change in response to specific molecules is reported. Stimuli‐responsiveness is imparted to ELMs by integrating genetically engineered yeast that only proliferate in the presence of specific biomolecules. This proliferation, in turn, leads to a shape change in the ELM in response to that biomolecule. These ELMs are fabricated by coprinting bioinks that contain multiple yeast strains. Locally, cellular proliferation leads to controllable shape change of the material resulting in up to a 370% increase in volume. Globally, the printed 3D structures contain regions of material that increase in volume and regions that do not under a given set of conditions, leading to programmable changes in form in response to target amino acids and nucleotides. Finally, this printing method is applied to design a reservoir‐based drug delivery system for the on‐demand delivery of a model drug in response to a specific biomolecule.
more »
« less
Enhancing long‐term storage and stability of engineered living materials through desiccant storage and trehalose treatment
Abstract Engineered living materials (ELMs) have broad applications for enabling on‐demand bioproduction of compounds ranging from small molecules to large proteins. However, most formulations and reports lack the capacity for storage beyond a few months. In this study, we develop an optimized procedure to maximize stress resilience of yeast‐laden ELMs through the use of desiccant storage and 10% trehalose incubation before lyophilization. This approach led to over 1‐year room temperature storage stability across a range of strain genotypes. In particular, we highlight the superiority of exogenously added trehalose over endogenous, engineered production in yielding robust preservation resilience that is independent of cell state. This simple, effective protocol enables sufficient accumulation of intracellular trehalose over a short period of contact time across a range of strain backgrounds without requiring the overexpression of a trehalose importer. A variety of microscopic analysis including µ‐CT and confocal microscopy indicate that cells form spherical colonies within F127‐BUM ELMs that have variable viability upon storage. The robustness of the overall procedure developed here highlights the potential for widespread deployment to enable on‐demand, cold‐chain independent bioproduction.
more »
« less
- Award ID(s):
- 2029249
- PAR ID:
- 10390610
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Biotechnology and Bioengineering
- Volume:
- 120
- Issue:
- 2
- ISSN:
- 0006-3592
- Format(s):
- Medium: X Size: p. 572-582
- Size(s):
- p. 572-582
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract While the archival digital memory industry approaches its physical limits, the demand is significantly increasing, therefore alternatives emerge. Recent efforts have demonstrated DNA’s enormous potential as a digital storage medium with superior information durability, capacity, and energy consumption. However, the majority of the proposed systems require on-demand de-novo DNA synthesis techniques that produce a large amount of toxic waste and therefore are not industrially scalable and environmentally friendly. Inspired by the architecture of semiconductor memory devices and recent developments in gene editing, we created a molecular digital data storage system called “DNA Mutational Overwriting Storage” (DMOS) that stores information by leveraging combinatorial, addressable, orthogonal, and independent in vitro CRISPR base-editing reactions to write data on a blank pool of greenly synthesized DNA tapes. As a proof of concept, this work illustrates writing and accurately reading of both a bitmap representation of our school’s logo and the title of this study on the DNA tapes.more » « less
-
Background:Adenosine triphosphate (ATP) levels guide many aspects of the red blood cell (RBC) hypothermic storage lesions. As a result, efforts to improve the quality of hypothermic-stored red cell concentrates (RCCs) have largely centered around designing storage solutions to promote ATP retention. Considering reduced temperatures alone would diminish metabolism, and thereby enhance ATP retention, we evaluated: (a) whether the quality of stored blood is improved at −4°C relative to conventional 4°C storage, and (b) whether the addition of trehalose and PEG400 can enhance these improvements. Study Design and Methods:Ten CPD/SAGM leukoreduced RCCs were pooled, split, and resuspended in a next-generation storage solution (i.e., PAG3M) supplemented with 0–165 mM of trehalose or 0–165 mM of PEG400. In a separate subset of samples, mannitol was removed at equimolar concentrations to achieve a fixed osmolarity between the additive and non-additive groups. All samples were stored at both 4°C and −4°C under a layer of paraffin oil to prevent ice formation. Results:PEG400 reduced hemolysis and increased deformability in −4°C-stored samples when used at a concentration of 110 mM. Reduced temperatures did indeed enhance ATP retention; however, in the absence of an additive, the characteristic storage-dependent decline in deformability and increase in hemolysis was exacerbated. The addition of trehalose enhanced this decline in deformability and hemolysis at −4°C; although, this was marginally alleviated by the osmolarity-adjustments. In contrast, outcomes with PEG400 were worsened by these osmolarity adjustments, but at no concentration, in the absence of these adjustments, was damage greater than the control. Discussion:Supercooled temperatures can allow for improved ATP retention; however, this does not translate into improved storage success. Additional work is necessary to further elucidate the mechanism of injury that progresses at these temperatures such that storage solutions can be designed which allow RBCs to benefit from this diminished rate of metabolic deterioration. The present study suggests that PEG400 could be an ideal component in these solutions.more » « less
-
Abstract Engineered living materials (ELMs) are a fast-growing area of research that combine approaches in synthetic biology and material science. Here, we engineer B. subtilis to become a living component of a silica material composed of self-assembling protein scaffolds for functionalization and cross-linking of cells. B. subtilis is engineered to display SpyTags on polar flagella for cell attachment to SpyCatcher modified secreted scaffolds. We engineer endospore limited B. subtilis cells to become a structural component of the material with spores for long-term storage of genetic programming. Silica biomineralization peptides are screened and scaffolds designed for silica polymerization to fabricate biocomposite materials with enhanced mechanical properties. We show that the resulting ELM can be regenerated from a piece of cell containing silica material and that new functions can be incorporated by co-cultivation of engineered B. subtilis strains. We believe that this work will serve as a framework for the future design of resilient ELMs.more » « less
-
Abstract Engineered living materials (ELMs) are an emerging class of biohybrid materials with genetically programmable functionalities. Integrating ELMs with 3D bioprinting synergizes their biological programmability with the geometry‐driven functionality of 3D‐printed constructs, transforming these materials into practical products and engineering solutions. This integration also introduces a new paradigm in additive manufacturing that harnesses the “livingness” of encapsulated microorganisms as an active element in the fabrication process to create adaptive and evolving 3D constructs. This Perspective presents recent advances in 3D bioprinting and discusses current developments at the intersection of 3D bioprinting and ELMs. It highlights opportunities at the interface of these two emerging fields, including understanding the interactions between living and nonliving components of ELMs for bioink design, incorporating synthetic biology into bioprinting workflows, utilizing microbial growth as a postprinting fabrication process, and integrating shape‐morphing materials to enable the 4D printing of ELMs.more » « less
