Cynthia H. Collins

A synthetic multicellular system for programmed pattern formation.

Pattern formation is a hallmark of coordinated cell behaviour in both single and multicellular organisms. It typically involves cell–cell communication and intracellular signal processing. Here we show a synthetic multicellular system in which genetically engineered ‘receiver’ cells are programmed to form ring-like patterns of differentiation based on chemical gradients of an acyl-homoserine lactone (AHL) signal that is synthesized by ‘sender’ cells. In receiver cells, ‘band-detect’ gene networks respond to user-defined ranges of AHL concentrations. By fusing different fluorescent proteins as outputs of network variants, an initially undifferentiated ‘lawn’ of receivers is engineered to form a bullseye pattern around a sender colony. Other patterns, such as ellipses and clovers, are achieved by placing senders in different configurations. Experimental and theoretical analyses reveal which kinetic parameters most significantly affect ring development over time. Construction and study of such synthetic multicellular systems can improve our quantitative understanding of naturally occurring developmental processes and may foster applications in tissue engineering, biomaterial fabrication and biosensing.

A synthetic Escherichia coli predator–prey ecosystem

We have constructed a synthetic ecosystem consisting of two Escherichia coli populations, which communicate bi‐directionally through quorum sensing and regulate each others gene expression and survival via engineered gene circuits. Our synthetic ecosystem resembles canonical predator–prey systems in terms of logic and dynamics. The predator cells kill the prey by inducing expression of a killer protein in the prey, while the prey rescue the predators by eliciting expression of an antidote protein in the predator. Extinction, coexistence and oscillatory dynamics of the predator and prey populations are possible depending on the operating conditions as experimentally validated by long‐term culturing of the system in microchemostats. A simple mathematical model is developed to capture these system dynamics. Coherent interplay between experiments and mathematical analysis enables exploration of the dynamics of interacting populations in a predictable manner.

Modular optimization of multi-gene pathways for fatty acids production in E. coli

Microbial fatty acid-derived fuels have emerged as promising alternatives to petroleum-based transportation fuels. Here we report a modular engineering approach that systematically removed metabolic pathway bottlenecks and led to significant titre improvements in a multi-gene fatty acid metabolic pathway. On the basis of central pathway architecture, E. coli fatty acid biosynthesis was re-cast into three modules: the upstream acetyl coenzyme A formation module; the intermediary acetyl-CoA activation module; and the downstream fatty acid synthase module. Combinatorial optimization of transcriptional levels of these three modules led to the identification of conditions that balance the supply of acetyl-CoA and consumption of malonyl-CoA/ACP. Refining protein translation efficiency by customizing ribosome binding sites for both the upstream acetyl coenzyme A formation and fatty acid synthase modules enabled further production improvement. Fed-batch cultivation of the engineered strain resulted in a final fatty acid production of 8.6 g l(-1). The modular engineering strategies demonstrate a generalized approach to engineering cell factories for valuable metabolites production.

Dual selection enhances the signaling specificity of a variant of the quorum-sensing transcriptional activator LuxR

The transcription factor LuxR activates gene expression in response to binding the signaling molecule 3-oxo-hexanoyl-homoserine lactone (3OC6HSL), an acyl-HSL with a carbonyl substituent at the third carbon of the acyl chain. We previously described a LuxR variant, LuxR-G2E, that activates gene expression by binding a broader range of acyl-HSLs, including straight-chain acyl-HSLs to which LuxR does not respond. Here, we use a dual positive-negative selection system to identify a variant of LuxR-G2E that retains the response to straight-chain acyl-HSLs, but no longer responds to 3OC6HSL. A single mutation, R67M, reduces LuxR-G2Es response to acyl-HSLs having a carbonyl substituent at the third carbon of the acyl chain. This specificity-enhancing mutation would not have been identified by positive selection alone. The dual selection system provides a rapid and reliable method for identifying LuxR variants that have or lack the desired response to a given set of acyl-HSL signals. LuxR variants with altered signaling specificities might become useful components for constructing artificial cell-cell communication systems that program population level behaviors.

Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones.

LuxR‐type  transcriptional  regulators  play  key  roles in quorum‐sensing systems that employ acyl‐homoserine lactones (acyl‐HSLs) as signal molecules. These proteins mediate quorum control by changing their interactions with RNA polymerase and DNA in response to binding their cognate acyl‐HSL. The evolutionarily related LuxR‐type proteins exhibit considerable diversity in primary sequence and in their response to acyl‐HSLs having acyl groups of differing length and composition. Little is known about which residues determine acyl‐HSL specificity, and less about the evolutionary time scales required to forge new ones. To begin to examine such issues, we have focused on the LuxR protein from Vibrio fischeri, which activates gene transcription in response to binding its cognate quorum signal, 3‐oxohexanoyl‐homoserine lactone (3OC6HSL). Libraries of luxR mutants were screened for variants exhibiting increased gene activation in response to octanoyl‐HSL (C8HSL), with which wild‐type LuxR interacts only weakly. Eight LuxR variants were identified that showed a 100‐fold increase in sensitivity to C8HSL; these variants also displayed increased sensitivities to pentanoyl‐HSL and tetradecanoyl‐HSL, while maintaining a wild‐type or greater response to 3OC6HSL. The most sensitive variants activated gene transcription as strongly with C8HSL as the wild type did with 3OC6HSL. With one exception, the amino acid residues involved were restricted to the N‐terminal, ‘signal‐binding’ domain of LuxR. These residue positions differed from critical positions previously identified via ‘loss‐of‐function’ mutagenesis. We have demonstrated that acyl‐HSL‐dependent quorum‐sensing systems can evolve rapidly to respond to new acyl‐HSLs, suggesting that there may be an evolutionary advantage to maintaining such plasticity.

Towards synthetic microbial consortia for bioprocessing.

The use of microbial consortia for bioprocessing has been limited by our ability to reliably control community composition and function simultaneously. Recent advances in synthetic biology have enabled population-level coordination and control of ecosystem stability and dynamics. Further, new experimental and computational tools for screening and predicting community behavior have also been developed. The integration of synthetic biology with metabolic engineering at the community level is vital to our ability to apply system-level approaches to building and optimizing synthetic consortia for bioprocessing applications. This review details new methods, tools and opportunities that together have the potential to enable a new paradigm of bioprocessing using synthetic microbial consortia.

Spaceflight Promotes Biofilm Formation by Pseudomonas aeruginosa

Understanding the effects of spaceflight on microbial communities is crucial for the success of long-term, manned space missions. Surface-associated bacterial communities, known as biofilms, were abundant on the Mir space station and continue to be a challenge on the International Space Station. The health and safety hazards linked to the development of biofilms are of particular concern due to the suppression of immune function observed during spaceflight. While planktonic cultures of microbes have indicated that spaceflight can lead to increases in growth and virulence, the effects of spaceflight on biofilm development and physiology remain unclear. To address this issue, Pseudomonas aeruginosa was cultured during two Space Shuttle Atlantis missions: STS-132 and STS-135, and the biofilms formed during spaceflight were characterized. Spaceflight was observed to increase the number of viable cells, biofilm biomass, and thickness relative to normal gravity controls. Moreover, the biofilms formed during spaceflight exhibited a column-and-canopy structure that has not been observed on Earth. The increase in the amount of biofilms and the formation of the novel architecture during spaceflight were observed to be independent of carbon source and phosphate concentrations in the media. However, flagella-driven motility was shown to be essential for the formation of this biofilm architecture during spaceflight. These findings represent the first evidence that spaceflight affects community-level behaviors of bacteria and highlight the importance of understanding how both harmful and beneficial human-microbe interactions may be altered during spaceflight.

Adenosine to inosine editing by ADAR2 requires formation of a ternary complex on the GluR-B R/G site.

RNA editing by members of the ADAR (adenosine deaminase that acts on RNA) enzyme family involves hydrolytic deamination of adenosine to inosine within the context of a double-stranded pre-mRNA substrate. Editing of the human GluR-B transcript is catalyzed by the enzyme ADAR2 at the Q/R and R/G sites. We have established a minimal RNA substrate for editing based on the R/G site and have characterized the interaction of ADAR2 with this RNA by gel shift, kinetic, and cross-linking analyses. Gel shift analysis revealed that two complexes are formed on the RNA as protein concentration is increased; the ADAR monomers can be cross-linked to one another in an RNA-dependent fashion. We performed a detailed kinetic study of the editing reaction; the data from this study are consistent with a reaction scheme in which formation of an ADAR2·RNA ternary complex is required for efficient RNA editing and in which formation of this complex is rate determining. These observations suggest that RNA adenosine deaminases function as homodimers on their RNA substrates and may partially explain regulation of RNA editing in these systems.

Engineering proteins that bind, move, make and break DNA

Recent protein engineering efforts have generated artificial transcription factors that bind new target DNA sequences and enzymes that modify DNA at new target sites. Zinc-finger-based transcription factors are favored targets for design; important technological advances in their construction and numerous biotechnological applications have been reported. Other notable advances include the generation of endonucleases and recombinases with altered specificities, made by innovative combinatorial and evolutionary protein engineering strategies. An unexpectedly high tolerance to mutation in the active sites of DNA polymerases is being exploited to engineer polymerases to incorporate artificial nucleotides or to display other, nonnatural activities.

Peptide-based communication system enables Escherichia coli to Bacillus megaterium interspecies signaling

The use of mixtures of microorganisms, or microbial consortia, has the potential to improve the productivity and efficiency of increasingly complex bioprocesses. However, the use of microbial consortia has been limited by our ability to control and coordinate the behaviors of microorganisms in synthetic communities. Synthetic biologists have previously engineered cell–cell communication systems that employ machinery from bacterial quorum‐sensing (QS) networks to enable population‐level control of gene expression. However, additional communication systems, such as those that enable communication between different species of bacteria, are needed to enable the use of diverse species in microbial consortia for bioprocessing. Here, we use the agr QS system from Staphylococcus aureus to generate an orthogonal synthetic communication system between Gram‐negative Escherichia coli and Gram‐positive Bacillus megaterium that is based on the production and recognition of autoinducing peptides (AIPs). We describe the construction and characterization of two types of B. megaterium “receiver” cells, capable of AIP‐dependent gene expression in response to AIPs that differ by a single amino acid. Further, we observed interspecies communication when these receiver cells were co‐cultured with AIP‐producing E. coli. We show that the two AIP‐based systems exhibit differences in sensitivity and specificity that may be advantageous in tuning communication‐dependent networks in synthetic consortia. These peptide‐based communication systems will enable the coordination of gene expression, metabolic pathways and growth between diverse microbial species, and represent a key step towards the use of microbial consortia in bioprocessing and biomanufacturing. Biotechnol. Bioeng. 2013;110: 3003–3012.