Cysteine Enhances Bioavailability of Copper to Marine Phytoplankton.

Emiliania huxleyi, a ubiquitous marine algae, was cultured under replete and Cu-limiting conditions to investigate Cu uptake strategies involving thiols and associated redox reactions; comparisons to a model diatom, Thalassiosira pseudonana, were also drawn. Cu-limitation increased rates of cell surface reduction of Cu(II) to Cu(I) in E. huxleyi but not in T. pseudonana. Furthermore, Cu-limited E. huxleyi cells took up more Cu when cysteine was present compared to when no ligand was added, although a dependence on cysteine concentration was not observed. In contrast, Cu uptake by replete cells was dependent upon the relative abundance of inorganic species [Cu(I)']. We also show that cysteine can increase the bioavailability of Cu to Cu-limited cells, of both species, through the reductive release of Cu(I) from fairly strong Cu(II) ligands such as EDTA. Finally, support for a mechanism involving uptake of a Cys-Cu complex in E. huxleyi is drawn from the observation that Cu-limitation significantly enhances cysteine uptake by transporters that exhibit Michaelis-Menten kinetics. These Cu uptake strategies help explain the presence and distribution of dissolved thiols in surface seawater and have implications for the biogeochemical cycling of Cu in low Cu environments.

Automated design of protein-binding riboswitches for sensing human biomarkers in a cell-free expression system.

Cell-free genetically encoded biosensors have been developed to detect small molecules and nucleic acids, but they have yet to be reliably engineered to detect proteins. Here we develop an automated platform to convert protein-binding RNA aptamers into riboswitch sensors that operate within low-cost cell-free assays. We demonstrate the platform by engineering 35 protein-sensing riboswitches for human monomeric C-reactive protein, human interleukin-32γ, and phage MS2 coat protein. The riboswitch sensors regulate output expression levels by up to 16-fold with input protein concentrations within the human serum range. We identify two distinct mechanisms governing riboswitch-mediated regulation of translation rates and leverage computational analysis to refine the protein-binding aptamer regions, improving design accuracy. Overall, we expand the cell-free sensor toolbox and demonstrate how computational design is used to develop protein-sensing riboswitches with future applications as low-cost medical diagnostics.

Tuning Cell-Free Composition Controls the Time Delay, Dynamics, and Productivity of TX-TL Expression.

The composition of cell-free expression systems (TX-TL) is adjusted by adding macromolecular crowding agents and salts. However, the effects of these cosolutes on the dynamics of individual gene expression processes have not been quantified. Here, we carry out kinetic mRNA and protein level measurements on libraries of genetic constructs using the common cosolutes PEG-8000, Ficoll-400, and magnesium glutamate. By combining these measurements with biophysical modeling, we show that cosolutes have differing effects on transcription initiation, translation initiation, and translation elongation rates with trade-offs between time delays, expression tunability, and maximum expression productivity. We also confirm that biophysical models can predict translation initiation rates in TX-TL using Escherichia coli lysate. We discuss how cosolute composition can be tuned to maximize performance across different cell-free applications, including biosensing, diagnostics, and biomanufacturing.

Purification of Cas9-RNA complexes by ultrafiltration.

The discovery of CRISPR-Cas9 has revolutionized molecular biology, greatly accelerating the introduction of genetic modifications into organisms and facilitating the development of novel therapeutics and diagnostics. For many applications, guide RNA and Cas9 protein are expressed, combined, and purified to produce a ribonucleic enzyme complex that is then added into a diagnostic device or delivered into cells. The objective of this work was to develop an ultrafiltration process for the selective purification of Cas9 ribonucleoprotein by removal of excess guide RNA. A His-tagged Streptococcus pyogenes Cas9 protein was produced in Escherichia coli, purified by metal affinity chromatography, and complexed with a 40 kDa (124 nucleotide) single guide RNA. Ultrafiltration experiments were first performed on solutions containing either guide RNA or Cas9 protein to identify the effect of filtration conditions and membrane pore size on the selectivity. Shear-induced aggregation of the Cas9 led to significant fouling under some conditions. A diafiltration process was then developed using a Biomax® 300 kDa polyethersulfone membrane to selectively remove excess guide RNA from a solution containing Cas9-bound guide RNA and free guide RNA. These results demonstrate the potential of using ultrafiltration for the removal of excess RNA during the production of functional ribonucleoprotein complexes.

Simultaneous repression of multiple bacterial genes using nonrepetitive extra-long sgRNA arrays.

Engineering cellular phenotypes often requires the regulation of many genes. When using CRISPR interference, coexpressing many single-guide RNAs (sgRNAs) triggers genetic instability and phenotype loss, due to the presence of repetitive DNA sequences. We stably coexpressed 22 sgRNAs within nonrepetitive extra-long sgRNA arrays (ELSAs) to simultaneously repress up to 13 genes by up to 3,500-fold. We applied biophysical modeling, biochemical characterization and machine learning to develop toolboxes of nonrepetitive genetic parts, including 28 sgRNA handles that bind Cas9. We designed ELSAs by combining nonrepetitive genetic parts according to algorithmic rules quantifying DNA synthesis complexity, sgRNA expression, sgRNA targeting and genetic stability. Using ELSAs, we created three highly selective phenotypes in Escherichia coli, including redirecting metabolism to increase succinic acid production by 150-fold, knocking down amino acid biosynthesis to create a multi-auxotrophic strain and repressing stress responses to reduce persister cell formation by 21-fold. ELSAs enable simultaneous and stable regulation of many genes for metabolic engineering and synthetic biology applications.