DNA Microgels as a Platform for Cell-Free Protein Expression and Display.

Protein expression and selection is an essential process in the modification of biological products. Expressed proteins are selected based on desired traits (phenotypes) from diverse gene libraries (genotypes), whose size may be limited due to the difficulties inherent in diverse cell preparation. In addition, not all genes can be expressed in cells, and linking genotype with phenotype further presents a great challenge in protein engineering. We present a DNA gel-based platform that demonstrates the versatility of two DNA microgel formats to address fundamental challenges of protein engineering, including high protein yield, isolation of gene sets, and protein display. We utilize microgels to show successful protein production and capture of a model protein, green fluorescent protein (GFP), which is further used to demonstrate a successful gene enrichment through fluorescence-activated cell sorting (FACS) of a mixed population of microgels containing the GFP gene. Through psoralen cross-linking of the hydrogels, we have synthesized DNA microgels capable of surviving denaturing conditions while still possessing the ability to produce protein. Lastly, we demonstrate a method of producing extremely high local gene concentrations of up to 32 000 gene repeats in hydrogels 1 to 2 µm in diameter. These DNA gels can serve as a novel cell-free platform for integrated protein expression and display, which can be applied toward more powerful, scalable protein engineering and cell-free synthetic biology with no physiological boundaries and limitations.

Standardization of functional reporter and antibiotic resistance cassettes to facilitate the genetic engineering of filamentous fungi.

The unique physiological properties of fungi are useful for a myriad of applications, which could greatly benefit from increased control of native pathways and introduction of recombinant genes. However, fungal genetic engineering is still limited in scope and accessibility, largely due to lack of standardization. To help standardize the genetic engineering of filamentous fungi, we created BioBricks of commonly used antibiotic resistance genes, neomycin phosphotransferase (nptII) and hygromycin phosphotransferase (hph), which confer resistance to G418 (Geneticin) and hygromycin B, respectively. Additionally, we created a BioBrick of the constitutive trpC promoter, from the tryptophan biosynthesis pathway of Aspergillus nidulans, and used it to create a composite part including the GFP gene. The functionality of these parts was demonstrated in the model fungal organism Cochliobolus heterostrophus, and as these tools are in modular BioBrick format, they can be easily used to facilitate genetic engineering of other fungal species.

An arsenic-specific biosensor with genetically engineered Shewanella oneidensis in a bioelectrochemical system.

Genetically engineered microbial biosensors have yet to realize commercial success in environmental applications due, in part, to difficulties associated with transducing and transmitting traditional bioluminescent information. Bioelectrochemical systems (BESs) output a direct electric signal that can be incorporated into devices for remote environmental monitoring. Here, we describe a BES-based biosensor with genetically encoded specificity for a toxic metal. By placing an essential component of the metal reduction (Mtr) pathway of Shewanella oneidensis under the control of an arsenic-sensitive promoter, we have genetically engineered a strain that produces increased current in response to arsenic when inoculated into a BES. Our BES-based biosensor has a detection limit of ~40 µM arsenite with a linear range up to 100 µM arsenite. Because our transcriptional circuit relies on the activation of a single promoter, similar sensing systems may be developed to detect other analytes by the swap of a single genetic part.