XPORT ENTRAP: A droplet microfluidic platform for enhanced DNA transfer between microbial species.

A significant hurdle for the widespread implementation and use of synthetic biology is the challenge of highly efficient introduction of DNA into microorganisms. This is especially a barrier for the utilization of non-model organisms and/or novel chassis species for a variety of applications, ranging from molecular biology to biotechnology and biomanufacturing applications. Common approaches to episomal and chromosomal gene editing, which employ techniques such as chemical competence and electroporation, are typically only amenable to a small subset of microbial species while leaving the vast majority of microorganisms in nature genetically inaccessible. To address this challenge, we have employed the previously described B. subtilis broad-host conjugation strain, XPORT, which was modularly designed for loading DNA cargo and conjugating such DNA into recalcitrant microbes. In this current work, we have leveraged and adapted the XPORT strain for use in a droplet microfluidic platform to enable increased efficiency of conjugation-based DNA transfer. The system named DNA ENTRAP (DNA ENhanced TRAnsfer Platform) utilizes cell-encapsulated water-in-oil emulsion droplets as pico-liter-volume bioreactors that allows controlled contacts between the donor and receiver cells within the emulsion bioreactor. This allowed enhanced XPORT-mediated genetic transfer over the current benchtop XPORT process, demonstrated using two different Bacillus subtilis strains (donor and receiver), as well as increased throughput (e.g., number of successfully conjugated cells) due to the automated assay steps inherent to microfluidic lab-on-a-chip systems. DNA ENTRAP paves the way for a streamlined automation of culturing and XPORT-mediated genetic transfer processes as well as future high-throughput cell engineering and screening applications.

The next generation of biopanning: next gen sequencing improves analysis of bacterial display libraries.

BACKGROUND: Bacterial surface display libraries are a popular tool for novel ligand discovery due to their ease of manipulation and rapid growth rates. These libraries typically express a scaffold protein embedded within the outer membrane with a short, surface-exposed peptide that is either terminal or is incorporated into an outer loop, and can therefore interact with and bind to substrates of interest. RESULTS: In this study, we employed a novel bacterial peptide display library which incorporates short 15-mer peptides on the surface of E. coli, co-expressed with the inducible red fluorescent protein DsRed in the cytosol, to investigate population diversity over two rounds of biopanning. The naive library was used in panning trials to select for binding affinity against 3D printing plastic coupons made from polylactic acid (PLA). Resulting libraries were then deep-sequenced using next generation sequencing (NGS) to investigate selection and diversity. CONCLUSIONS: We demonstrated enrichment for PLA binding versus a sapphire control surface, analyzed population composition, and compared sorting rounds using a binding assay and fluorescence microscopy. The capability to produce and describe display libraries through NGS across rounds of selection allows a deeper understanding of population dynamics that can be better directed towards peptide discovery.

Engineered integrative and conjugative elements for efficient and inducible DNA transfer to undomesticated bacteria.

Engineering microorganisms to promote human or plant health will require manipulation of robust bacteria that are capable of surviving in harsh, competitive environments. Genetic engineering of undomesticated bacteria can be limited by an inability to transfer DNA into the cell. Here we developed an approach based on the integrative and conjugative element from Bacillus subtilis (ICEBs1) to overcome this problem. A donor strain (XPORT) was built to transfer miniaturized integrative and conjugative elements (mini-ICEBs1) to undomesticated bacteria. The strain was engineered to enable inducible control over conjugation, to integrate delivered DNA into the chromosome of the recipient, to restrict spread of heterologous DNA through separation of the type IV secretion system from the transferred DNA, and to enable simple isolation of engineered bacteria through a D-alanine auxotrophy. Efficient DNA transfer (10-1 to 10-7 conjugation events per donor) is demonstrated using 35 Gram-positive strains isolated from humans (skin and gut) and soil. Mini-ICEBs1 was used to rapidly characterize the performance of an isopropyl-ß-D-thiogalactoside (IPTG)-inducible reporter across dozens of strains and to transfer nitrogen fixation to four Bacillus species. Finally, XPORT was introduced to soil to demonstrate DNA transfer under non-ideal conditions.

The Next Generation of Synthetic Biology Chassis: Moving Synthetic Biology from the Laboratory to the Field.

Escherichia coli (E. coli) has played a pivotal role in the development of genetics and molecular biology as scientific fields. It is therefore not surprising that synthetic biology (SB) was built upon E. coli and continues to dominate the field. However, scientific capabilities have advanced from simple gene mutations to the insertion of rationally designed, complex synthetic circuits and creation of entirely synthetic genomes. The point is rapidly approaching where E. coli is no longer an adequate host for the increasingly sophisticated genetic designs of SB. It is time to develop the next generation of SB chassis; robust organisms that can provide the advanced physiology novel synthetic circuits will require to move SB from the laboratory into fieldable technologies. This can be accomplished by developing chassis-specific genetic toolkits that are as extensive as those for E. coli. However, the holy grail of SB would be the development of a universal toolkit that can be ported into any chassis. This viewpoint article underscores the need for new bacterial chassis, as well as discusses some of the important considerations in their selection. It also highlights a few examples of robust, tractable bacterial species that can meet the demands of tomorrow's state-of-the-art in SB. Significant advances have been made in the first 15 years since this field has emerged. However, the advances over the next 15 years will occur not in laboratory organisms, but in fieldable species where the potential of SB can be fully realized in game changing technology.

Evolved Quorum sensing regulator, LsrR, for altered switching functions.

In order to carry out innovative complex, multistep synthetic biology functions, members of a cell population often must communicate with one another to coordinate processes in a programmed manner. It therefore follows that native microbial communication systems are a conspicuous target for developing engineered populations and networks. Quorum sensing (QS) is a highly conserved mechanism of bacterial cell-cell communication and QS-based synthetic signal transduction pathways represent a new generation of biotechnology toolbox members. Specifically, the E. coli QS master regulator, LsrR, is uniquely positioned to actuate gene expression in response to a QS signal. In order to expand the use of LsrR in synthetic biology, two novel LsrR switches were generated through directed evolution: an "enhanced" repression and derepression eLsrR and a reversed repression/derepression function "activator" aLsrR. Protein modeling and docking studies are presented to gain insight into the QS signal binding to these two evolved proteins and their newly acquired functionality. We demonstrated the use of the aLsrR switch using a coculture system in which a QS signal, produced by one bacterial strain, is used to inhibit gene expression via aLsrR in a different strain. These first ever AI-2 controlled synthetic switches allow gene expression from the lsr promoter to be tuned simultaneously in two distinct cell populations. This work expands the tools available to create engineered microbial populations capable of carrying out complex functions necessary for the development of advanced synthetic products.

Genetically engineered peptides for inorganics: study of an unconstrained bacterial display technology and bulk aluminum alloy.

The first-ever peptide biomaterial discovery using an unconstrained engineered bacterial display technology is reported. Using this approach, we have developed genetically engineered peptide binders for a bulk aluminum alloy and use molecular dynamics simulation of peptide conformational fluctuations to demonstrate sequence-dependent, structure-function relationships for metal and metal oxide interactions.

Developing next generation antimicrobials by intercepting AI-2 mediated quorum sensing.

Bacteria have been evolving antibiotic resistance since their discovery in the early twentieth century. Most new antibiotics are derivatives of older generations and there are now bacteria that are virtually resistant to almost all antibiotics. This poses a global threat to human health and has been classified as a clinical "super-challenge", which has necessitated research into new antimicrobials that inhibit bacterial virulence while minimizing selective pressures that lead to the emergence of resistant strains. Quorum sensing (QS), the process of population dependent bacterial cell-cell signaling, can accelerate bacterial virulence and is an increasingly interesting target for developing next generation antimicrobials. Most QS inhibitors target species-specific signals, such as acylhomoserine lactones (AHLs) and oligopeptides. Methodologies for intercepting the cross-species signal, autoinducer-2 (AI-2), have only recently emerged. We review these strategies to prevent the relay of the AI-2 signal amongst pathogens, including Escherichia coli, Salmonella enterica serovar Typhimurium, Vibrio cholerae and Pseudomonas aeruginosa. Inhibition mechanisms are categorized based on the target (i.e., enzymes for signal generation, the signal molecule itself, or the various components of the signal transduction process). The universal nature of the AI-2 signal imparts on its inhibitors the potential for broad spectrum use.

Bio-tarp alternative daily cover prototypes for methane oxidation atop open landfill cells.

Final landfill covers are highly engineered to prevent methane release into the atmosphere. However, methane production begins soon after waste placement and is an unaddressed source of emissions. The methane oxidation capacity of methanotrophs embedded in a "bio-tarp" was investigated as a means to mitigate methane release from open landfill cells. The bio-tarp would also serve as an alternative daily cover during routine landfill operation. Evaluations of nine synthetic geotextiles identified two that would likely be suitable bio-tarp components. Pilot tarp prototypes were tested in continuous flow systems simulating landfill gas conditions. Multilayered bio-tarp prototypes consisting of alternating layers of the two geotextiles were found to remove 16% of the methane flowing through the bio-tarp. The addition of landfill cover soil, compost, or shale amendments to the bio-tarp increased the methane removal up to 32%. With evidence of methane removal in a laboratory bioreactor, prototypes were evaluated at a local landfill using flux chambers installed atop intermediate cover at a landfill. The multilayered bio-tarp and amended bio-tarp configurations were all found to decrease landfill methane flux; however, the performance efficacy of bio-tarps was not significantly different from controls without methanotrophs. Because highly variable methane fluxes at the field site likely confounded the test results, repeat field testing is recommended under more controlled flux conditions.