bioRxiv Science⌕ Search

bioRxiv · 10.1101/756049

Experimental Evidence for Constraints in Amplitude-Timescale Co-variation of a Biomolecular Pulse Generating Circuit Design

Abstract

Understanding constraints on the functional properties of biomolecular circuit dynamics, such as the variation of amplitude and timescale of pulse, is an important part of biomolecular circuit design. While the amplitude-timescale co-variations of the pulse in an incoherent feedforward loop have been investigated computationally using mathematical models, experimental support for such constraints is relatively unclear. Here, we address this using experimental measurements of an existing pulse generating incoherent feedforward loop circuit realization in the context of a standard mathematical model. We characterize the trends of co-variation in the pulse amplitude and rise time computationally by randomly exploring the parameter space. We experimentally measured the co-variation by varying inducers and found that larger amplitude pulses have slower rise time. We discuss the gap between the experimental measurements and predictions of the standard model, highlighting model additions and other biological factors that might bridge the gap.

Source connections

Explore related subjects

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Patel, A., Sen, S.. 2019-09-03. Experimental Evidence for Constraints in Amplitude-Timescale Co-variation of a Biomolecular Pulse Generating Circuit Design. https://doi.org/10.1101/756049

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

Machine learning guided design of strong transcriptional activation domains

Artificial intelligence has accelerated the design of structured proteins, but it remains difficult to design functional intrinsically disordered proteins because of weak structural constraints and poorly defined sequence-to-function relationships1. Activation domains (ADs), transcription factor regions that recruit coactivators, are an excellent test case for the de novo design of intrinsically disordered sequences. ADs are one of the oldest groups of functional intrinsically disordered sequences2 and designed ADs have applications in synthetic biology, biotechnology, and agriculture. We computationally generated 500,000 candidate sequences that resembled known ADs and predicted AD function with extant machine learning models, including three neural networks. From high-scoring sequences, we selected 18,750 for experimental validation in yeast. Surprisingly, >90% of the 16,770 well-measured sequences function as ADs. Sequences predicted to be active by multiple models were stronger, motivating development of a fast and accurate consensus AD predictor, MADRIGAL. A number of our designed ADs retain functionality across fungi, plants, and human cells. This work illustrates a generalizable paradigm for incorporating sequence-to-function models into the design loop to efficiently generate functional intrinsically disordered sequences.

synthetic biology↗

E. coli-produced nanobody fusions as a pan-serotype platform for AAV purification, quantification, and detection

Research, production and application of the recombinant adeno-associated virus (rAAV) require versatile tools for rapid and cost-effective advances. We developed fusion constructs with the pan-serotype, AAV-binding VHH-antibody-fragment named nanobody AAVX and devised procedures for AAV purification, quantification, and detection. Starting from an amino acid sequence, we cytosolically expressed in E. coli nanobody AAVX carrying various tags and fusion partners. Very efficient soluble production followed by a one-step immobilized metal-ion affinity chromatography (IMAC) yielded mostly >90% pure protein. Crosslinking AAVX with an agarose matrix enabled efficient purification of intact AAV2 and AAV5 capsids from cell lysates, as assessed by qPCR and ELISA. AAVX also supported effective AAV quantification by ELISA, as capture reagent and, when fused to luciferase, as detection reagent. Using tagged AAVX and streptavidin biosensors, we developed a rapid and label-free quantification assay of AAV capsids by biolayer interferometry (BLI). Finally, immunostaining with an AAVX-eGFP fusion visualized AAV capsids in fixed HEK-293 cells by fluorescence microscopy. Collectively, these results establish nanobody AAVX fusion proteins as versatile and readily customizable tools for AAV research and bioprocessing.

synthetic biology↗

Duality Vaccination through Nanodesign of Click-Ligated B-Cell Inhibitory Receptor Bridging

Synthesis of precise and robust molecular architectures is critical to harness the power of the immune system. The dimensions, rigidity and multivalency of nanoparticles contribute to eliciting high levels of antibodies from vaccination. However, antibodies vary hugely in quality: some antibodies are highly effective at neutralizing pathogens, while others provide little or no benefit. Here we establish switchable and modular platforms for protein and nanoparticle assembly that program the target of antibody responses. We introduce Duality vaccination: the simultaneous delivery of activating and inhibitory stimuli for focusing immune responses. To construct activatory immunogens, we employ click biology with spontaneous amidation to functionalize plug-and-display self-assembling porous dodecahedral nanocages. We construct inhibitory immunogens using SpyMask protease-gated bispecific assembly, ligating the antigen to binders that bridge the B cell receptor to immune checkpoint receptors. This dual immunogen architecture tailors the elicited antibodies to down-modulate undesired B cell responses. We establish the effect of temporal and spatial separation of activatory and inhibitory immunogen delivery. We further explore sensitivity to valency, linker flexibility and adjuvant delivery, employing model targets and coronavirus antigens. Dual programmable functionalization identifies key nanodesign features to control in vivo outputs, providing logic-gating towards urgent challenges in the creation of protective vaccines.

synthetic biology↗