Compartmentalized Cell-Free Expression Systems for Building Synthetic Cells.

One of the grand challenges in bottom-up synthetic biology is the design and construction of synthetic cellular systems. One strategy toward this goal is the systematic reconstitution of biological processes using purified or non-living molecular components to recreate specific cellular functions such as metabolism, intercellular communication, signal transduction, and growth and division. Cell-free expression systems (CFES) are in vitro reconstitutions of the transcription and translation machinery found in cells and are a key technology for bottom-up synthetic biology. The open and simplified reaction environment of CFES has helped researchers discover fundamental concepts in the molecular biology of the cell. In recent decades, there has been a drive to encapsulate CFES reactions into cell-like compartments with the aim of building synthetic cells and multicellular systems. In this chapter, we discuss recent progress in compartmentalizing CFES to build simple and minimal models of biological processes that can help provide a better understanding of the process of self-assembly in molecularly complex systems.

A Multiplexed Cas13-Based Assay with Point-of-Care Attributes for Simultaneous COVID-19 Diagnosis and Variant Surveillance.

Point-of-care (POC) nucleic acid detection technologies are poised to aid gold-standard technologies in controlling the COVID-19 pandemic, yet shortcomings in the capability to perform critically needed complex detection-such as multiplexed detection for viral variant surveillance-may limit their widespread adoption. Herein, we developed a robust multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)-based detection using LwaCas13a and PsmCas13b to simultaneously diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and pinpoint the causative SARS-CoV-2 variant of concern (VOC)-including globally dominant VOCs Delta (B.1.617.2) and Omicron (B.1.1.529)-all the while maintaining high levels of accuracy upon the detection of multiple SARS-CoV-2 gene targets. The platform has several attributes suitable for POC use: premixed, freeze-dried reagents for easy use and storage; convenient direct-to-eye or smartphone-based readouts; and a one-pot variant of the multiplexed detection. To reduce reliance on proprietary reagents and enable sustainable use of such a technology in low- and middle-income countries, we locally produced and formulated our own recombinase polymerase amplification reaction and demonstrated its equivalent efficiency to commercial counterparts. Our tool-CRISPR-based detection for simultaneous COVID-19 diagnosis and variant surveillance that can be locally manufactured-may enable sustainable use of CRISPR diagnostics technologies for COVID-19 and other diseases in POC settings.

Discovery and Genetic Code Expansion of a Polyethylene Terephthalate (PET) Hydrolase from the Human Saliva Metagenome for the Degradation and Bio-Functionalization of PET.

We report a bioinformatic workflow and subsequent discovery of a new polyethylene terephthalate (PET) hydrolase, which we named MG8, from the human saliva metagenome. MG8 has robust PET plastic degradation activities under different temperature and salinity conditions, outperforming several naturally occurring and engineered hydrolases in degrading PET. Moreover, we genetically encoded 2,3-diaminopropionic acid (DAP) in place of the catalytic serine residue of MG8, thereby converting a PET hydrolase into a covalent binder for bio-functionalization of PET. We show that MG8(DAP), in conjunction with a split green fluorescent protein system, can be used to attach protein cargos to PET as well as other polyester plastics. The discovery of a highly active PET hydrolase from the human metagenome-currently an underexplored resource for industrial enzyme discovery-as well as the repurposing of such an enzyme into a plastic functionalization tool, should facilitate ongoing efforts to degrade and maximize reusability of PET.

Molecular probes for cellular imaging of post-translational proteoforms.

Specific post-translational modification (PTM) states of a protein affect its property and function; understanding their dynamics in cells would provide deep insight into diverse signaling pathways and biological processes. However, it is not trivial to visualize post-translational modifications in a protein- and site-specific manner, especially in a living-cell context. Herein, we review recent advances in the development of molecular imaging tools to detect diverse classes of post-translational proteoforms in individual cells, and their applications in studying precise roles of PTMs in regulating the function of cellular proteins.

Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA.

Nucleic acid detection by isothermal amplification and the collateral cleavage of reporter molecules by CRISPR-associated enzymes is a promising alternative to quantitative PCR. Here, we report the clinical validation of the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) assay using the enzyme Cas13a from Leptotrichia wadei for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the virus that causes coronavirus disease 2019 (COVID-19)-in 154 nasopharyngeal and throat swab samples collected at Siriraj Hospital, Thailand. Within a detection limit of 42 RNA copies per reaction, SHERLOCK was 100% specific and 100% sensitive with a fluorescence readout, and 100% specific and 97% sensitive with a lateral-flow readout. For the full range of viral load in the clinical samples, the fluorescence readout was 100% specific and 96% sensitive. For 380 SARS-CoV-2-negative pre-operative samples from patients undergoing surgery, SHERLOCK was in 100% agreement with quantitative PCR with reverse transcription. The assay, which we show is amenable to multiplexed detection in a single lateral-flow strip incorporating an internal control for ribonuclease contamination, should facilitate SARS-CoV-2 detection in settings with limited resources.

Rapid Detection of the Antibiotic Sulfamethazine in Pig Body Fluids by Paper Spray Mass Spectrometry.

We report herein a practical method for nonlethal detection of the antibiotic sulfamethazine in pig body fluids via the combination of simple extraction and paper spray mass spectrometry (PS-MS). This method requires minimal sample preparation while still providing high sensitivities and accuracies in complex matrices including pig whole blood (LOD = 7.9 µg/L; recovery = 95.4-103.7%), pig serum (LOD = 11.5 µg/L; recovery = 103.2-106.2%), and synthetic urine (LOD = 11.2 µg/L; recovery = 99.1-103.2%). Given a known correlation between the level of sulfamethazine in body fluids and edible tissues, this method shows great promise as a practical and nonlethal solution for rapid testing of the drug, which can substantially aid managerial decision in the livestock industry.