Exogenous pathway optimization and chassis engineering are two crucial methods for heterologous pathway expression. The two methods are normally carried out step-wise and in a trial-and-error manner. Here we report a recombinase-based combinatorial method (termed “SCRaMbLE-in”) to tackle both challenges simultaneously. SCRaMbLE-in includes an in vitro recombinase toolkit to rapidly prototype and diversify gene expression at the pathway level and an in vivo genome reshuffling system to integrate assembled pathways into the synthetic yeast genome while combinatorially causing massive genome rearrangements in the host chassis. A set of loxP mutant pairs was identified to maximize the efficiency of the in vitro diversification. Exemplar pathways of β-carotene and violacein were successfully assembled, diversified, and integrated using this SCRaMbLE-in method. High-throughput sequencing was performed on selected engineered strains to reveal the resulting genotype-to-phenotype relationships. The SCRaMbLE-in method proves to be a rapid, efficient, and universal method to fast track the cycle of engineering biology.

Sensitive detection of protein interactions and post-translational modifications of native proteins is a challenge for research and diagnostic purposes. A method for this, which could be used in point-of-care devices and high-throughput screening, should be reliable, cost effective and robust. To achieve this, here we design a method (proxHCR) that combines the need for proximal binding with hybridization chain reaction (HCR) for signal amplification. When two oligonucleotide hairpins conjugated to antibodies bind in close proximity, they can be activated to reveal an initiator sequence. This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product. In conclusion, we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry. As no enzymes are needed, proxHCR may be an inexpensive and robust alternative to proximity ligation assays.

Play it again, SAM. Peptide substrates immobilised on self‐assembled monolayer (SAM)‐coated gold surfaces were efficiently glycosylated with UDP‐N‐acetyl‐α‐D‐galactosamine:polypeptide N‐acetylgalactosaminyltransferase 2 (ppGalNAcT2) to form the α‐D‐GalNAc threonine/serine linkage. Label‐free analysis of the reactions were performed by using MALDI‐ToF mass spectrometry.

We study phase separation in suspensions of two unrelated species of rod-like bacteria, Escherichia coli and Sinorhizobium meliloti, induced by the addition of two different anionic polyelectrolytes, sodium polystyrene sulfonate or succinoglycan, the former being synthetic and the latter of natural origin. Comparison with the known behaviour of synthetic colloid-polymer mixtures and with simulations show that "depletion" (or, equivalently, "macromolecular crowding") is the dominant mechanism: exclusion of the non-adsorbing polymer from the region between two neighbouring bacteria creates an unbalanced osmotic force pushing them together. The implications of our results for understanding phenomena such as biofilm formation are discussed.

We studied aggregation and phase separation in suspensions of de-flagellated Escherichia coli (AB1157) in phosphate buffer induced by the anionic polyelectrolyte sodium polystyrene sulfonate. We also performed Monte Carlo simulations of this system based on the Asakura-Oosawa model of colloidpolymer mixtures. The results of these simulations, as well as comparison with previous work on synthetic colloid-polymer mixtures, demonstrate that the role of the polymer is to cause a depletion attraction between the E. coli cells. The implication of these results for understanding the role of (predominantly anionic) extracellular polymeric substances (EPS) secreted by bacteria in various natural phenomena such as biofilm formation is discussed.