Biotechnology software in the digital age: are you winning?

There is a digital revolution taking place and biotechnology companies are slow to adapt. Many pharmaceutical, biotechnology, and industrial bio-production companies believe that software must be developed and maintained in-house and that data are more secure on internal servers than on the cloud. In fact, most companies in this space continue to employ large IT and software teams and acquire computational infrastructure in the form of in-house servers. This is due to a fear of the cloud not sufficiently protecting in-house resources and the belief that their software is valuable IP. Over the next decade, the ability to quickly adapt to changing market conditions, with agile software teams, will quickly become a compelling competitive advantage. Biotechnology companies that do not adopt the new regime may lose on key business metrics such as return on invested capital, revenue, profitability, and eventually market share.

Wet Lab Accelerator: A Web-Based Application Democratizing Laboratory Automation for Synthetic Biology.

Wet Lab Accelerator (WLA) is a cloud-based tool that allows a scientist to conduct biology via robotic control without the need for any programming knowledge. A drag and drop interface provides a convenient and user-friendly method of generating biological protocols. Graphically developed protocols are turned into programmatic instruction lists required to conduct experiments at the cloud laboratory Transcriptic. Prior to the development of WLA, biologists were required to write in a programming language called "Autoprotocol" in order to work with Transcriptic. WLA relies on a new abstraction layer we call "Omniprotocol" to convert the graphical experimental description into lower level Autoprotocol language, which then directs robots at Transcriptic. While WLA has only been tested at Transcriptic, the conversion of graphically laid out experimental steps into Autoprotocol is generic, allowing extension of WLA into other cloud laboratories in the future. WLA hopes to democratize biology by bringing automation to general biologists.

Construction of a genetic multiplexer to toggle between chemosensory pathways in Escherichia coli.

Many applications require cells to switch between discrete phenotypic states. Here, we harness the FimBE inversion switch to flip a promoter, allowing expression to be toggled between two genes oriented in opposite directions. The response characteristics of the switch are characterized using two-color cytometry. This switch is used to toggle between orthogonal chemosensory pathways by controlling the expression of CheW and CheW*, which interact with the Tar (aspartate) and Tsr* (serine) chemoreceptors, respectively. CheW* and Tsr* each contain a mutation at their protein-protein interface such that they interact with each other. The complete genetic program containing an arabinose-inducible FimE controlling CheW/CheW* (and constitutively expressed tar/tsr*) is transformed into an Escherichia coli strain lacking all native chemoreceptors. This program enables bacteria to swim toward serine or aspartate in the absence or in the presence of arabinose, respectively. Thus, the program functions as a multiplexer with arabinose as the selector. This demonstrates the ability of synthetic genetic circuits to connect to a natural signaling network to switch between phenotypes.

Kinetic buffering of cross talk between bacterial two-component sensors.

Two-component systems are a class of sensors that enable bacteria to respond to environmental and cell-state signals. The canonical system consists of a membrane-bound sensor histidine kinase that autophosphorylates in response to a signal and transfers the phosphate to an intracellular response regulator. Bacteria typically have dozens of two-component systems. The key questions are whether these systems are linear and, if they are, how cross talk between systems is buffered. In this work, we studied the EnvZ/OmpR and CpxA/CpxR systems from Escherichia coli, which have been shown previously to exhibit slow cross talk in vitro. Using in vitro radiolabeling and a rapid quenched-flow apparatus, we experimentally measured 10 biochemical parameters capturing the cognate and non-cognate phosphotransfer reactions between the systems. These data were used to parameterize a mathematical model that was used to predict how cross talk is affected as different genes are knocked out. It was predicted that significant cross talk between EnvZ and CpxR only occurs for the triple mutant DeltaompR DeltacpxA DeltaactA-pta. All seven combinations of these knockouts were made to test this prediction and only the triple mutant demonstrated significant cross talk, where the cpxP promoter was induced 280-fold upon the activation of EnvZ. Furthermore, the behavior of the other knockouts agrees with the model predictions. These results support a kinetic model of buffering where both the cognate bifunctional phosphatase activity and the competition between regulator proteins for phosphate prevent cross talk in vivo.

The molecular basis of species-specific ligand activation of trace amine-associated receptor 1 (TAAR(1)).

The trace amine-associated receptor 1 (TAAR(1)) is an aminergic G protein-coupled receptor (GPCR) potently activated by 3-iodothyronamine (1), an endogenous derivative of thyroid hormone. Structure-activity relationship studies on 1 and related agonists showed that the rat and mouse species of TAAR(1) accommodated structural modifications and functional groups on the ethylamine portion and the biaryl ether moiety of the molecule. However, the two receptors clearly exhibited distinct, species-specific ligand preferences despite being remarkably similar with 93% sequence similarity. In this study, we generated single and double mutants of rat and mouse TAAR(1) to probe the molecular recognition of agonists and the underlying basis for the ligand selectivity of rat and mouse TAAR(1). Key, nonconserved specificity determinant residues in transmembranes helices 4 and 7 within the ligand binding site appear to be the primary source of a number of the observed ligand preferences. Residue 7.39 in transmembrane 7 dictated the preference for a beta-phenyl ring, while residue 4.56 in transmembrane 4 was partially responsible for the lower potency of 1 and tyramine for the mouse receptor. Additionally, 1 and tyramine were found to have the same binding mode in rat TAAR(1) despite structure-activity relationship data suggesting the possibility of each molecule having different binding orientations. These findings provide valuable insights into the critical binding site residues involved in the ligand-receptor interaction that can influence compound selectivity and functional activity of aminergic GPCRs.

Toward deciphering the code to aminergic G protein-coupled receptor drug design.

The trace amine-associated receptor 1 (TAAR(1)) is a biogenic amine G protein-coupled receptor (GPCR) that is potently activated by 3-iodothyronamine (1, T(1)AM) in vitro. Compound 1 is an endogenous derivative of the thyroid hormone thyroxine which rapidly induces hypothermia, anergia, and bradycardia when administered to mice. To explore the role of TAAR(1) in mediating the effects of 1, we rationally designed and synthesized rat TAAR(1) superagonists and lead antagonists using the rotamer toggle switch model of aminergic GPCR activation. The functional activity of a ligand is proposed to be correlated to its probable interactions with the rotamer switch residues; agonists allow the rotamer switch residues to toggle to their active conformation, whereas antagonists interfere with this conformational transition. These agonist and antagonist design principles provide a conceptual model for understanding the relationship between the molecular structure of a drug and its pharmacological properties.

Strengths of hydrogen bonds involving phosphorylated amino acid side chains.

Post-translational phosphorylation plays a key role in regulating protein function. Here, we provide a quantitative assessment of the relative strengths of hydrogen bonds involving phosphorylated amino acid side chains (pSer, pAsp) with several common donors (Arg, Lys, and backbone amide groups). We utilize multiple levels of theory, consisting of explicit solvent molecular dynamics, implicit solvent molecular mechanics, and quantum mechanics with a self-consistent reaction field treatment of solvent. Because the approximately 6 pKa of phosphate suggests that -1 and -2 charged species may coexist at physiological pH, hydrogen bonds involving both protonated and deprotonated phosphates for all donor-acceptor pairs are considered. Multiple bonding geometries for the charged-charged interactions are also considered. Arg is shown to be capable of substantially stronger salt bridges with phosphorylated side chains than Lys. A pSer hydrogen-bond acceptor tends to form more stable interactions than a pAsp acceptor. The effect of phosphate protonation state on the strengths of the hydrogen bonds is remarkably subtle, with a more pronounced effect on pAsp than on pSer.

Conformational changes in protein loops and helices induced by post-translational phosphorylation.

Post-translational phosphorylation is a ubiquitous mechanism for modulating protein activity and protein-protein interactions. In this work, we examine how phosphorylation can modulate the conformation of a protein by changing the energy landscape. We present a molecular mechanics method in which we phosphorylate proteins in silico and then predict how the conformation of the protein will change in response to phosphorylation. We apply this method to a test set comprised of proteins with both phosphorylated and non-phosphorylated crystal structures, and demonstrate that it is possible to predict localized phosphorylation-induced conformational changes, or the absence of conformational changes, with near-atomic accuracy in most cases. Examples of proteins used for testing our methods include kinases and prokaryotic response regulators. Through a detailed case study of cyclin-dependent kinase 2, we also illustrate how the computational methods can be used to provide new understanding of how phosphorylation drives conformational change, why substituting Glu or Asp for a phosphorylated amino acid does not always mimic the effects of phosphorylation, and how a phosphatase can "capture" a phosphorylated amino acid. This work illustrates how computational methods can be used to elucidate principles and mechanisms of post-translational phosphorylation, which can ultimately help to bridge the gap between the number of known sites of phosphorylation and the number of structures of phosphorylated proteins.

Binding of the Bacillus subtilis LexA protein to the SOS operator.

The Bacillus subtilis LexA protein represses the SOS response to DNA damage by binding as a dimer to the consensus operator sequence 5'-CGAACN(4)GTTCG-3'. To characterize the requirements for LexA binding to SOS operators, we determined the operator bases needed for site-specific binding as well as the LexA amino acids required for operator recognition. Using mobility shift assays to determine equilibrium constants for B.subtilis LexA binding to recA operator mutants, we found that several single base substitutions within the 14 bp recA operator sequence destabilized binding enough to abolish site-specific binding. Our results show that the AT base pairs at the third and fourth positions from the 5' end of a 7 bp half-site are essential and that the preferred binding site for a LexA dimer is 5'-CGAACATATGTTCG-3'. Binding studies with LexA mutants, in which the solvent accessible amino acid residues in the putative DNA binding domain were mutated, indicate that Arg-49 and His-46 are essential for binding and that Lys-53 and Ala-48 are also involved in operator recognition. Guided by our mutational analyses as well as hydroxyl radical footprinting studies of the dinC and recA operators we docked a computer model of B.subtilis LexA on the preferred operator sequence in silico. Our model suggests that binding by a LexA dimer involves bending of the DNA helix within the internal 4 bp of the operator.

Genetic composition of the Bacillus subtilis SOS system.

The SOS response in bacteria includes a global transcriptional response to DNA damage. DNA damage is sensed by the highly conserved recombination protein RecA, which facilitates inactivation of the transcriptional repressor LexA. Inactivation of LexA causes induction (derepression) of genes of the LexA regulon, many of which are involved in DNA repair and survival after DNA damage. To identify potential RecA-LexA-regulated genes in Bacillus subtilis, we searched the genome for putative LexA binding sites within 300 bp upstream of the start codons of all annotated open reading frames. We found 62 genes that could be regulated by putative LexA binding sites. Using mobility shift assays, we found that LexA binds specifically to DNA in the regulatory regions of 54 of these genes, which are organized in 34 putative operons. Using DNA microarray analyses, we found that 33 of the genes with LexA binding sites exhibit RecA-dependent induction by both mitomycin C and UV radiation. Among these 33 SOS genes, there are 22 distinct LexA binding sites preceding 18 putative operons. Alignment of the distinct LexA binding sites reveals an expanded consensus sequence for the B. subtilis operator: 5'-CGAACATATGTTCG-3'. Although the number of genes controlled by RecA and LexA in B. subtilis is similar to that of Escherichia coli, only eight B. subtilis RecA-dependent SOS genes have homologous counterparts in E. coli.