The current paradigm for tuning synthetic biological systems is through re-engineering system components. Biological systems designed with the inherent ability to be tuned by external stimuli will be more versatile. We engineered Escherichia coli cells to behave as an externally tunable band-pass filter for enzyme activity and small molecules. The band's location can be positioned within a range of 4 orders of magnitude simply by the addition of compounds to the growth medium. Inclusion in the genetic network of an enzyme-substrate pair that functions as an attenuator is a generalizable strategy that enables this tunability. The genetic circuit enabled bacteria growth to be patterned in response to chemical gradients in nonintuitive ways and facilitated the isolation of engineered allosteric enzymes. The application of this strategy to other biological systems will increase their utility for biotechnological applications and their usefulness as a tool for gaining insight into nature's underlying design principles.biological circuit ͉ biological engineering ͉ pattern formation ͉ protein switch ͉ synthetic biology E xternal control over cellular processes is invaluable for research and technology. For example, inducible promoters allow one to control the amount of gene transcription from the outside the cell, obviating the need to engineer different cell strains with different promoters for each level of transcription desired. Moreover, inducible promoters enable control of transcription levels as a function of time and position. They consist of a simple genetic circuit that has a set input-output relation between inducer concentration and transcription level. This input-output relation cannot be conveniently altered from outside the cell. Thus, although the inducer can be said to ''tune'' the level of transcription, the circuit itself is not externally tunable. To tune these circuits, researchers must modify system components (e.g., by changing a repressor's affinity for its inducer). This approach to tuning currently predominates in the design of synthetic biological systems. However, such an approach is tantamount to building a unique biological system for each desired behavior and precludes the possibility of dynamic tuning or spatially patterned tuning. For example, Basu et al.(1) created a multicellular system that functioned as a band-detect filter for a small molecule. They demonstrated how the position of the band could be shifted to different concentration ranges by changing a repressor's affinity for a signaling molecule and by altering gene copy number. Tuning was achieved through building a different biological system for each desired band position. However, electronic band-detect filters can be built such that any desired range can be set dynamically through adjusting knobs from outside the device. The ability to create analogous externally tuneable biological systems would greatly expand their versatility. We describe how proper inclusion of an enzymesubstrate pair in the network results in an exter...

BackgroundGradients of morphogens pattern cell fate – a phenomenon that is especially important during development. A simple model system for studying how morphogens pattern cell behavior would overcome difficulties inherent in the study of natural morphogens in vivo. A synthetic biology approach to building such a system is attractive.ResultsUsing an externally-tunable band-pass filter paradigm, we engineered Escherichia coli cells to function as a model system for the study of how multiple morphogens can pattern cell behavior. We demonstrate how our system exhibits behavior such as morphogen crosstalk and how the cells' growth and fluorescence can be patterned in a number of complex patterns. We extend our cell patterning from 2D cultures on the surface of plates to 3D cultures in soft agarose medium.ConclusionOur system offers a convenient, well-defined model system for fundamental studies on how multiple morphogen gradients can affect cell fate and lead to pattern formation. Our design principles could be applied to eukaryotic cells to develop other models systems for studying development or for enabling the patterning of cells for applications such as tissue engineering and biomaterials.

Regulation of protein activity is central to the complexity of life. The ability to regulate protein activity through exogenously added molecules has biotechnological/biomedical applications and offers tools for basic science. Such regulation can be achieved by establishing a means to modulate the specific activity of the protein (i.e. allostery). An alternative strategy for intracellular regulation of protein activity is to control the amount of protein through effects on its production, accumulation, and degradation. We have previously demonstrated that the non-homologous recombination of the genes encoding maltose binding protein (MBP) and TEM1 β-lactamase (BLA) can result in fusion proteins in which β-lactamase enzyme activity is allosterically regulated by maltose. Here, through use of a two-tiered genetic selection scheme, we demonstrate that such recombination can result in genes that confer maltose-dependent resistance to β-lactam even though they do not encode allosteric enzymes. These ‘phenotypic switch’ genes encode fusion proteins whose accumulation is a result of a specific interaction with maltose. Phenotypic switches represent an important class of proteins for basic science and biotechnological applications in vivo.

The capsular antigen detection (CAD) kit is widely used in clinics to detect Streptococcus pneumoniae infection from urine, because it is rapid, convenient, and effective. However, there are several disadvantages, including false-positive results in children colonized with S. pneumoniae and prolonged positive readings even after the bacteria have been cleared. RP-L7/L12 is a component of the 50S ribosome that is abundant in all bacteria and is specific for each bacterial species. We investigated whether RP-L7/L12 could be used to accurately diagnose pneumococcal pneumonia infection in mouse models of pneumonia and colonization generated by infecting CBA/JN or CBA/N mice, respectively, with S. pneumoniae strain 741. RP-L7/L12 detection by enzyme-linked immunosorbent assay accurately assessed active lung infection, as RP-L7/L12 levels decreased simultaneously with the bacterial lung burden after imipenem administration in the pneumonia mouse model. Based on the data, antibodies detecting RP-L7/L12 were applied to rapid immunochromatographic strips (ICS) for urine sample testing. When we compared the ICS test with the CAD kit in the pneumonia model, the results correlated well. Interestingly, however, when the lung bacterial burden became undetectable after antibiotic treatment, the ICS test was correspondingly negative, even though the same samples tested by the CAD kit remained positive. Similarly, while the ICS test exhibited negative results in the nasal colonization model, the CAD kit demonstrated positive results. Bacterial RP-L7/L12 may be a promising target for the development of new methods to diagnose infectious disease. Further studies are warranted to determine whether such a test could be useful in children.