Morphogen-defined patterning of Escherichia coli enabled by an externally tunable band-pass filter

<p>Abstract</p> <p>Background</p> <p>Gradients 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 <it>in vivo</it>. A synthetic biology approach to building such a system is attractive.</p> <p>Results</p> <p>Using an externally-tunable band-pass filter paradigm, we engineered <it>Escherichia coli </it>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.</p> <p>Conclusion</p> <p>Our 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.</p

Integrity testing of Planova™ BioEX virus removal filters used in the manufacture of biological products

AbstractConfirmation of virus filter integrity is crucial for ensuring the safety of biological products. Two main types of virus filter defects may produce inconsistent and undesirable performance in virus removal: improper pore-size distribution across the membrane; and specific damage, such as tears, broken fibers, or pinholes. Two integrity tests are performed on each individual filter manufactured by Asahi Kasei Medical to ensure the absence of these defects prior to shipment. In this study, we verified that typical usage of Planova™ BioEX filters would not improperly shift the pore-size distribution. Damage occurring during shipment and use (e.g., broken fibers or pinholes) can be detected by end-users with sufficient sensitivity using air–water diffusion based leakage tests. We prepared and tested filters with model pinhole defects of various sizes to develop standard acceptance criteria for the leakage test relative to porcine parvovirus infectivity logarithmic reduction values (LRVs). Our results demonstrate that pinhole defects at or below a certain size for each effective filter surface area have no significant impact on the virus LRV. In conclusion the leakage test is sufficiently sensitive to serve as the sole end-user integrity test for Planova™ BioEX filters, facilitating their use in biopharmaceuticals manufacturing

In Vitro Recombination of Non-Homologous Genes Can Result in Gene Fusions that Confer a Switching Phenotype to Cells

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

Sequence of fusion proteins.

a<p>Based on gene sequencing.</p

The effect of maltose on MIC_Amp and protein expression.

a<p>Protein expressed from plasmid pDIMC8 in RH22 cells.</p>b<p>MIC_Amp was determined in triplicate. For proteins with a MIC_Amp range, this indicates the range from the three experiments (e.g. Ph5 was found to have a MIC_Amp in the absence of maltose of 16 µg/ml in some trials and 32 µg/ml in other trials).</p>c<p>Accumulation ratio determined from quantitative image analysis of western blots probed using polyclonal anti-BLA antibodies (except for MBP which was probed with anti-MBP antibodies).</p>d<p>(With maltose)/(without maltose).</p>e<p>Standard deviation calculated from three independent experiments.</p>f<p>No pDIMC8 plasmid was present. The protein monitored by western blot was chromosomally encoded MBP.</p

Melting temperatures of proteins.

a<p>T_m, temperature at transition midpoint, determined by temperature-induced unfolding monitored by CD spectroscopy. All temperatures are ±0.2°C.</p

Effect of ligand concentration on MIC_Amp and accumulation of phenotypic switches.

<p>(A) Western blot using anti-BLA antibodies of the soluble fraction of cells expressing MBP317-347, Ph8 and sucrose switch 5–7 (arrow, ∼70 kDa). Cells were cultured with maltose (mal), sucrose (suc), or neither sugar (−). (B) Correlation between the MIC_Amp and the concentration of maltose added to the culture for cell expressing MBP317-347 (•) or MBP317-347(W340A) (□). (C) Accumulation levels dictate the MIC_Amp. Correlation between the accumulation ratio (+maltose/−maltose), as measured by western blot, and the MIC_Amp ratio (+maltose/−maltose) of cell expressing MBP317-347 (•) or MBP317-347(W340A) (□). Different accumulation ratios were obtained by culturing the cells in the presence of different concentrations of maltose.</p

Kinetic constants for the hydrolysis of ampicillin^a.

a<p>Assays were at 37°C in 10 mM phosphate buffer (pH 7.0) with or without 5 mM maltose.</p>b<p>At 30°C in 50 mM phosphate buffer (pH 7.0) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027302#pone.0027302-Raquet1" target="_blank">[9]</a>. Previous studies have shown that BLA enzyme activity is unaffected by maltose <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027302#pone.0027302-Guntas3" target="_blank">[4]</a>.</p>c<p>(<i>k</i>_cat/K_m)_+maltose/(<i>k</i>_cat/K_m)_−maltose.</p