Synthetic Biology

eLife assessment

This study presents a useful investigation to test de novo-designed mini binders against the Spike protein of SARS-CoV-2 within two classes of synthetic receptors (SNIPRs and CARs). The methods and evidence supporting the focused claims are very solid, although the small-scale nature of the investigation (number of modifications, number of minibinders, etc.) makes it difficult to determine how generalizable these results and potential design principles are. This work will be of interest to synthetic biologists and cell engineers as a starting point for systematic, larger-scale analysis and optimization of synthetic receptor designs for cellular therapy and other applications.

eLife assessment

The manuscript describes a valuable method to boost WNT signaling in a tissue-specific manner. The work extends previous data from the authors based on fusing an RSPO2 mutant protein to an antibody that binds ASGR1/2. In the current manuscript, two new antibodies with similar effects are described, that expand this solid approach and provide alternatives for potential future clinical applications. This manuscript will be of interest to all scientists studying protein engineering and cellular targeting.

eLife assessment

This important study uses an innovative set of reporter assays to probe the role of the TnpB protein in IS608 transposition. The work provides independent support for the recently reported homing activity of TnpB, where the transposon is restored following excision, and suggests an additional function for TnpB in enhancing the transposase activity of the TnpA transposase. The overall approach is solid, but the authors should consider how the activity of the TnpB protein used, or the levels of ωRNA, impact their model.

eLife Assessment

This important study demonstrates the use of the mammalian Musashi-1 (MSI-1) RNA-binding protein as a tool for regulating gene expression in Escherichia coli. The authors provide convincing evidence that MSI-1 functions as an effective repressor of translation, and that MSI-1 can be allosterically controlled by oleic acid. This work establishes MSI-1 as a potential tool for synthetic biology applications, and the system developed here can be used for mechanistic studies of MSI-1.

eLife assessment

This important study presents a new way to selectively activate a cell signaling pathway in a specific cell type by designer ligands that link signaling co-receptors to a marker specific to the target cells. Convincing experimental results demonstrate that the agonist molecules activate Wnt signaling in target cells expressing the marker as intended. More broadly, this concept could be used to induce Wnt signaling or another pathway initiated by co-receptor association in a cell type-specific manner. In vitro results in this study could be further strengthened by assessing the biological consequences of Wnt activation in target cells.

eLife assessment

This valuable study focuses on the impact of growth feedback on the performance of artificial gene circuits capable of achieving adaptive responses, an important problem in synthetic biology. Through solid computational analysis, the authors identify specific failure mechanisms, as well as core topologies associated with robust performance based on systematic analysis of over four hundred circuit topologies. The results will be of interest to those working on engineering gene circuits for diverse applications.

eLife assessment

The authors aim to develop a CRISPR system that can be activated upon sensing an RNA. As an initial step to this goal, they describe RNA-sensing guide RNAs for controlled activation of CRISPR modification. Many of the data look convincing and while several steps remain to achieve the stated goal in an in vivo setting and for robust activation by endogenous RNAs, the current work will be important for many in the field.

eLife assessment

This is an important follow-up study to a previous paper in which the authors reconstituted CO2 metabolism (autotrophy) in Escherichia coli. Here, the authors define a set of just three mutations that promote autotrophy, highlighting the malleability of E. coli metabolism. The authors make a convincing case that mutations in pgi are loss-of-function mutations that prevent metabolic efflux from the reductive pentose phosphate autocatalytic cycle, and their data suggest possible roles of mutations in two other genes - crp and rpoB. This research will be particularly interesting to synthetic biologists, systems biologists, and metabolic engineers aiming to develop synthetic autotrophic microorganisms.

eLife assessment

This study presents a valuable and comprehensive mutagenesis map of the AAV2 rep gene, which will undoubtedly capture the interest of scientists working with adeno-associated viruses and those engaged in the field of gene therapy. The thorough characterization of massive rep variants across multiple AAV production systems bolsters the claims made in the study, highlighting its utility in enhancing our understanding of Rep protein function and advancing gene therapy applications. The evidence presented is convincing and establishes a strong foundation that will stimulate and inform future research in the field.

eLife assessment

This valuable study reports on a new tool that allows for light-controlled protein degradation in Escherichia coli. With the improved light-responsive protein tag, endogenous protein levels can be reduced severalfold. The methodology is convincing and will be of interest to the fields of gene expression regulation in bacteria and, more generally to synthetic biologists.

eLife assessment

Experimental models of simple cell-like compartments can help us to understand how biology operated early in its history. The authors convincingly show how the properties of coacervate droplets can be influenced by the activity of ribozymes inside them. This important result potentially provides a new route for biologists or chemists to establish cell mimics that support the evolution of biomolecules within.

eLife assessment

This manuscript will be of interest to those working on non-neuronal bioelectricity, particular synthetic biologists and bioengineers. The primary contribution is the ability to leverage engineered gene circuits to control cellular membrane potential. We find issue, however, with the presentation of the data in this work as electrical communication since the synchronous behavior largely arises from external chemical stimuli.

Evaluation Summary:

In this study, Trolle et al aimed to introduce methionine, threonine, isoleucine, and valine biosynthetic pathways into Chinese Hamster Ovary (CHO) cells. While this was unsuccessful for methionine, threonine, and isoleucine, introduction of valine synthesis rendered CHO cells partially independent on exogenous valine. Although introduction of essential amino acid biosynthetic pathways into mammalian cells is of potentially broad interest to the fields of synthetic biology, biotechnology and metabolism, there were concerns regarding incomplete demonstration that the introduction of valine pathway into CHO cells is sufficient to sustain homeostasis in the absence of exogenous valine. Further metabolic/biochemical characterization of valine-producing CHO cells is warranted.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

Evaluation Summary:

This is an interesting paper that uses de novo protein design to probe the effects of oligomerization state on the activity of chimeric antigen receptors (CARS). The successful design of transmembrane domains with specific oligomeric states is an impressive result on its own. The proteins were designed using rotamer-based sequence optimization in Rosetta with an energy function specific for the membrane environment. After experimentally evaluating a couple rounds of designs, the investigators settled on a design protocol that also included screening of the design candidates with docking simulations in alternative oligomerization states to check that the sequences preferred the desired oligomerization state. The designs were experimentally evaluated with gel electrophoresis and X-ray crystallography. In the end, designs that adopted well-defined dimers, trimers, or tetramers were created and carried forward in experiments as CARs.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

Evaluation Summary:

This study describes a novel approach to investigate how the transcriptional repressors KRAB and HDAC4 repress gene expression, how repression spreads over differing genomic distances, and what the role of insulator elements is in blocking the spread of repression and in reactivation of repressed genes. The results of this study allow modeling of the coordinated repression or activation of closely linked genes and should be of wide interest to researchers interested in chromatin and gene expression.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

Evaluation Summary:

Using a phage-based library generation and selection, the authors generated a suite of 4-base decoding tRNAs with improved efficiency in quadruplet decoding. The data represent an important step toward enhancing protein synthesis with 4-base codons. Overall, the approach to generate many tRNA variants with quadruplet anticodons is intriguing and provides a wealth of valuable information to the field. The results, once some of the reviewer concerns have been addressed, should become foundational for the field of synthetic biology.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

Evaluation Summary:

Challa and Ryu et al. systematically evaluated various combinations of ADP-ribose-binding modules to make sensors detecting poly(ADP-ribose). They developed and tested two indicator designs optimized for analyses in cell culture (dimerization-dependent GFP-based) or intact tissues (split Nano luciferase-based). Overall, with further experimental controls and quantification, this timely set of cell biology probes will be useful to study the biological functions of ADP-ribosylation in cultured cells and whole organisms.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

Evaluation Summary:

The manuscript dives into how protein structure/function robustness to mutation or polymorphism relates across evolutionary distance. The work indicates that evolutionarily related genes will have different shapes of robustness to variation, and that this will not necessarily track with phylogenetic relationships. The conclusions have potential ramifications for protein engineering, protein structure as well as population genetics and phylogenetics.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Evaluation Summary:

Three of Nature's life-sustaining processes, respiration, photosynthesis, and nitrogen fixation, all rely on proteins (Fe-S protein) that contain simple inorganic cofactors constructed of Fe and S (Fe-S clusters). Fe-S proteins also participate in a huge and diverse array of metabolic processes. As such there has been considerable interest over the past two decades towards understanding how Fe-S clusters are formed and distributed to their cognate proteins. A related issue, the topic of the present work, is: why is it that many Fe-S proteins from diverse microbial species cannot be heterologously produced in Escherichia coli in active forms? This issue is of considerable interest not only from the perspective of microbial Fe-S proteins but also for heterologous expression of active eukaryotic Fe-S proteins. The study provides insights on the phylogenetic and biosynthetic limitations concerning formation of functional heterologously expressed Fe-S proteins.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

Evaluation Summary:

In this manuscript, the authors address important problems in the field of synthetic biology about scalability, robustness, and modularity. They used multiple strains to build gene circuits and demonstrate the modular composition of strain circuits with an automated design strategy to achieve a target behavior from a large space of possible functional circuit architectures. The major claims of the manuscript are well supported by solid quantitative data and systematic mathematical modeling analysis, and the approaches used are thoughtful and rigorous. This paper is of interest to synthetic biologists within the field of designing community-level behaviors, such as distributed computing, in multicellular consortia.

(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)