Insightful directed evolution of Escherichia coli quorum sensing promoter region of the lsrACDBFG operon: a tool for synthetic biology systems and protein expression.

Quorum sensing (QS) regulates many natural phenotypes (e.q. virulence, biofilm formation, antibiotic resistance), and its components, when incorporated into synthetic genetic circuits, enable user-directed phenotypes. We created a library of Escherichia coli lsr operon promoters using error-prone PCR (ePCR) and selected for promoters that provided E. coli with higher tetracycline resistance over the native promoter when placed upstream of the tet(C) gene. Among the fourteen clones identified, we found several mutations in the binding sites of QS repressor, LsrR. Using site-directed mutagenesis we restored all p-lsrR-box sites to the native sequence in order to maintain LsrR repression of the promoter, preserving the other mutations for analysis. Two promoter variants, EP01rec and EP14rec, were discovered exhibiting enhanced protein expression. In turn, these variants retained their ability to exhibit the LsrR-mediated QS switching activity. Their sequences suggest regulatory linkage between CytR (CRP repressor) and LsrR. These promoters improve upon the native system and exhibit advantages over synthetic QS promoters previously reported. Incorporation of these promoters will facilitate future applications of QS-regulation in synthetic biology and metabolic engineering.

Bacteria Floc, but Do They Flock? Insights from Population Interaction Models of Quorum Sensing.

Quorum sensing (QS) enables coordinated, population-wide behavior. QS-active bacteria "communicate" their number density using autoinducers which they synthesize, collect, and interpret. Tangentially, chemotactic bacteria migrate, seeking out nutrients and other molecules. It has long been hypothesized that bacterial behaviors, such as chemotaxis, were the primordial progenitors of complex behaviors of higher-order organisms. Recently, QS was linked to chemotaxis, yet the notion that these behaviors can together contribute to higher-order behaviors has not been shown. Here, we mathematically link flocking behavior, commonly observed in fish and birds, to bacterial chemotaxis and QS by constructing a phenomenological model of population-scale QS-mediated phenomena. Specifically, we recast a previously developed mathematical model of flocking and found that simulated bacterial behaviors aligned well with well-known QS behaviors. This relatively simple system of ordinary differential equations affords analytical analysis of asymptotic behavior and describes cell position and velocity, QS-mediated protein expression, and the surrounding concentrations of an autoinducer. Further, heuristic explorations of the model revealed that the emergence of "migratory" subpopulations occurs only when chemotaxis is directly linked to QS. That is, behaviors were simulated when chemotaxis was coupled to QS and when not. When coupled, the bacterial flocking model predicts the formation of two distinct groups of cells migrating at different speeds in their journey toward an attractant. This is qualitatively similar to phenomena spotted in our Escherichiacoli chemotaxis experiments as well as in analogous work observed over 50 years ago.IMPORTANCE Our modeling efforts show how cell density can affect chemotaxis; they help to explain the roots of subgroup formation in bacterial populations. Our work also reinforces the notion that bacterial mechanisms are at times exhibited in higher-order organisms.

Stress Fracture of the Hamate Hook in a Water Polo Player.

Hamate hook fractures are usually caused by direct trauma while using a tennis racket or a baseball bat. We report stress fracture of the hamate hook in a water polo player without any specific trauma. We consider that the stress fracture occurred via indirect mechanisms through the flexor tendons. Strong ulnar deviation of the wrist during ball release and strong grip on a ball with outstretched fingers, which are unique to water polo, were the likely causes of the stress fracture of the hamate hook.

TumbleScore: Run and tumble analysis for low frame-rate motility videos.

Scientists often exploit the motility of peritrichously flagellated bacteria for various applications. A common alteration is modifying the frequency of mid-movement changes in direction, known as tumbles. Such differences in bacterial swimming patterns can prove difficult to quantify, especially for those without access to high-speed optical equipment. Traditionally, scientists have resorted to less accurate techniques, such as soft agar plate assays, or have been forced to invest in costly equipment. Here, we present TumbleScore, software designed to track and quantify bacterial movies with slow, as well as fast, frame-rates. Developed and fully contained within MATLAB, TumbleScore processes motility videos and returns pertinent tumbling metrics, including: (i) linear speed, (ii) rotational speed, (iii) percentage of angle changes below a given threshold, and (iv) ratio of total path length to Euclidian distance, or arc-chord ratio (ACR). In addition, TumbleScore produces a "rose graph" visualization of bacterial paths. The software was validated using both fabricated and experimental motility videos.

Electronic control of gene expression and cell behaviour in Escherichia coli through redox signalling.

The ability to interconvert information between electronic and ionic modalities has transformed our ability to record and actuate biological function. Synthetic biology offers the potential to expand communication 'bandwidth' by using biomolecules and providing electrochemical access to redox-based cell signals and behaviours. While engineered cells have transmitted molecular information to electronic devices, the potential for bidirectional communication stands largely untapped. Here we present a simple electrogenetic device that uses redox biomolecules to carry electronic information to engineered bacterial cells in order to control transcription from a simple synthetic gene circuit. Electronic actuation of the native transcriptional regulator SoxR and transcription from the PsoxS promoter allows cell response that is quick, reversible and dependent on the amplitude and frequency of the imposed electronic signals. Further, induction of bacterial motility and population based cell-to-cell communication demonstrates the versatility of our approach and potential to drive intricate biological behaviours.

Electrochemical Measurement of the ß-Galactosidase Reporter from Live Cells: A Comparison to the Miller Assay.

In order to match our ability to conceive of and construct cells with enhanced function, we must concomitantly develop facile, real-time methods for elucidating performance. With these, new designs can be tested in silico and steps in construction incrementally validated. Electrochemical monitoring offers the above advantages largely because signal transduction stems from direct electron transfer, allowing for potentially quicker and more integrated measurements. One of the most common genetic reporters, ß-galactosidase, can be measured both spectrophotometrically (Miller assay) and electrochemically. However, since the relationship between the two is not well understood, the electrochemical methods have not yet garnered the attention of biologists. With the aim of demonstrating the utility of an electrochemical measurement to the synthetic biology community, we created a genetic construct that interprets and reports (with ß-galactosidase) on the concentration of the bacterial quorum sensing molecule autoinducer-2. In this work, we provide a correlation between electrochemical measurements and Miller Units. We show that the electrochemical assay works with both lysed and whole cells, allowing for the prediction of one from the other, and for continuous monitoring of cell response. We further present a conceptually simple and generalized mathematical model for cell-based ß-galactosidase reporter systems that could aid in building and predicting a variety of synthetic biology constructs. This first-ever in-depth comparison and analysis aims to facilitate the use of electrochemical real-time monitoring in the field of synthetic biology as well as to facilitate the creation of constructs that can more easily communicate information to electronic systems.