Small ubiquitin-like modifier (SUMO) E3 ligases are known to have a major role in preventing gross chromosomal rearrangements (GCRs); however, relatively little is known about the role of SUMO isopeptidases in genome maintenance and their role in controlling intracellular sumoylation homeostasis. Here we show the SUMO isopeptidase Ulp2 in Saccharomyces cerevisiae does not prevent the accumulation of GCRs, and interestingly, its loss causes subunit-specific changes of sumoylated minichromosome maintenance (MCM) helicase in addition to drastic accumulation of sumoylated nucleolar RENT and inner kinetochore complexes. In contrast, loss of Ulp1 or its mis-localization from the nuclear periphery causes substantial accumulations of GCRs and elevated sumoylation of most proteins except for Ulp2 targets. Interestingly, the E3 ligase Mms21, which has a major role in genome maintenance, preferentially controls the sumoylation of Mcm3 during DNA replication. These findings reveal distinct roles for Ulp1 and Ulp2 in controlling homeostasis of intracellular sumoylation and show that sumoylation of MCM is controlled in a subunit-specific and cell cycle dependent manner.Protein sumoylation is an essential post-translational modification in eukaryotes (1, 2). Two families of enzymes control reversible sumoylation of specific substrates, including SUMO 3 (small ubiquitin-like modifier) E3 ligases and SUMO isopeptidases. Three SUMO E3 ligases Siz1, Siz2, and Mms21 have been identified in Saccharomyces cerevisiae and are shown to have distinct, but partially overlapping roles in catalyzing substratespecific sumoylation (3-6). Siz1 and Siz2 are paralogs, and they redundantly catalyze the bulk of sumoylation in cells (5, 6). Mms21 catalyzes sumoylation of fewer substrates but plays a more important role in genome maintenance than Siz1 and Siz2 (6, 7). Deletion of SIZ1 and SIZ2 is lethal in cells lacking Mms21 E3 ligase activity (4, 5). Moreover, deletion of either SIZ1 or SIZ2 causes further accumulation of gross chromosome rearrangements (GCRs) in cells lacking Mms21 E3 ligase activity (6). These findings suggest that the functions of these E3 ligases are partially redundant, which correlates with their partially overlapping roles in catalyzing intracellular sumoylation (5, 6).Besides SUMO E3 ligases, homeostasis of intracellular sumoylation is also regulated by SUMO isopeptidases, which catalyze the removal of SUMO from its targets. Two SUMO isopeptidases Ulp1 and Ulp2 have been identified in S. cerevisiae (8 -10). Ulp2 is not required for cell viability; however, its loss causes accumulation of poly-SUMO chains, resulting in pleiotropic effects including slow growth and sensitivity to higher temperature (9, 11). Moreover, overexpression of ULP2

Synthetic cells can mimic the intricate complexities of live cells, while mitigating the level of noise that is present natural systems; however, many crucial processes still need to be demonstrated in synthetic cells to use them to comprehensively study and engineer biology. Here we demonstrate key functionalities of synthetic cells previously available only to natural life: differentiation and mating. This work presents a toolset for engineering combinatorial genetic circuits in synthetic cells. We demonstrate how progenitor populations can differentiate into new lineages in response to small molecule stimuli or as a result of fusion, and we provide practical demonstration of utility for metabolic engineering. This work provides a tool for bioengineering and for natural pathway studies, as well as paving the way toward the construction of live artificial cells.

Building a live cell from non‐living building blocks would be a fundamental breakthrough in biological sciences, and it would enable engineering new lineages of life, not directly descendant of the Last Universal Common Ancestor. Fully engineered synthetic cells will have architectures that can be radically different from the natural cells, yet most life processes reconstituted in synthetic cells so far are built from natural and biosimilar building blocks. Most natural processes have already been reconstituted in synthetic cell chassis. This paper summarizes recent advancements in using non‐living building blocks to reconstitute some of the most crucial features of living systems in a fully engineerable chassis of a synthetic cell.

Isothermal, cell-free, synthetic biology-based approaches to pathogen detection leverage the power of tools available in biological systems, such as highly active polymerases compatible with lyophilization, without the complexity inherent to live-cell systems, of which nucleic acid sequence based amplification (NASBA) is well known. Despite the reduced complexity associated with cell-free systems, side reactions are a common characteristic of these systems. As a result, these systems often exhibit false positives from reactions lacking an amplicon. Here we show that the inclusion of a DNA duplex lacking a promoter and unassociated with the amplicon fully suppresses false positives, enabling a suite of fluorescent aptamers to be used as NASBA tags (Apta-NASBA). Apta-NASBA has a 1 pM detection limit and can provide multiplexed, multicolor fluorescent readout. Furthermore, Apta-NASBA can be performed using a variety of equipment, for example, a fluorescence microplate reader, a qPCR instrument, or an ultra-low-cost Raspberry Pi-based 3D-printed detection platform using a cell phone camera module, compatible with field detection.

Background Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. In addition, the yields of transcription in cell-free systems lag behind transcriptional efficiency of live cells. Most commonly used in vitro translation systems utilize T7 RNA polymerase, which is also the enzyme included in many commercial kits. Results Here we present characterization of a variant of T7 RNA polymerase promoter that acts to significantly increase the yields of gene expression within in vitro systems. We have demonstrated that T7Max increases the yield of translation in many types of commonly used in vitro protein expression systems. We also demonstrated increased protein expression yields from linear templates, allowing the use of T7Max driven expression from linear templates. Conclusions The modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase- based in vitro protein expression system.