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Adenylate kinases (AKs) have evolved AMP-binding and lid domains that are encoded as continuous polypeptides embedded at different locations within the discontinuous polypeptide encoding the core domain. A prior study showed that AK homologues of different stabilities consistently retain cellular activity following circular permutation that splits a region with high energetic frustration within the AMP-binding domain into discontinuous fragments. Herein, we show that mesophilic and thermophilic AKs having this topological restructuring retain activity and substrate-binding characteristics of the parental AK. While permutation decreased the activity of both AK homologues at physiological temperatures, the catalytic activity of the thermophilic AK increased upon permutation when assayed >30 °C below the melting temperature of the native AK. The thermostabilities of the permuted AKs were uniformly lower than those of native AKs, and they exhibited multiphasic unfolding transitions, unlike the native AKs, which presented cooperative thermal unfolding. In addition, proteolytic digestion revealed that permutation destabilized each AK in differing manners, and mass spectrometry suggested that the new termini within the AMP-binding domain were responsible for the increased proteolysis sensitivity. These findings illustrate how changes in contact order can be used to tune enzyme activity and alter folding dynamics in multidomain enzymes. 

Optogenetics is a powerful tool for spatiotemporal control of gene expression. Several light-inducible gene regulators have been developed to function in bacteria, and these regulatory circuits have been ported to new host strains. Here, we developed and adapted a red-light-inducible transcription factor for Shewanella oneidensis. This regulatory circuit is based on the iLight optogenetic system, which controls gene expression using red light. A thermodynamic model and promoter engineering were used to adapt this system to achieve differential gene expression in light and dark conditions within a S. oneidensis host strain. We further improved the iLight optogenetic system by adding a repressor to invert the genetic circuit and activate gene expression under red light illumination. The inverted iLight genetic circuit was used to control extracellular electron transfer within S. oneidensis. The ability to use both red- and blue-light-induced optogenetic circuits simultaneously was also demonstrated. Our work expands the synthetic biology capabilities in S. oneidensis, which could facilitate future advances in applications with electrogenic bacteria. 

About the slide set The slides are divided into sections (“Concepts”) including: What is engineering/synthetic biology? (Concept 1-1); the “Design, Build, Test, & Learn” cycle (Concept 1-2), Core Tools for engineering biology (Concepts 1-3 and 1-4), and finally exploring Impacts & Applications of engineering biology (Concept 1-5). The slides can be used as a complete lecture, or any concept topic can be used to supplement existing material. For example, Concept 1-1 could be used to introduce synthetic biology to professionals outside the field, or the Concept 1-5 Data Science section could be modified to show the intersections of the field in a computer science course. The slides are available to use under Creative Commons license CC BY-NC-SA. The goal of these slides is to provide free, accessible, and modular explanations of key Engineering Biology topics. EBRC provides this curricular module under a Creative Commons Attribution-NonCommercial-ShareAlike 2.0 license, which allows free use in noncommercial settings with credit for the material given to EBRC and the content authors. By downloading these resources, you agree to these terms. If you are interested in using EBRC material in a commercial setting or have other usage questions, please contact us at education@ebrc.org. The slides were created by Michael Sheets (Boston University) and Joshua Atkinson (Univ. of Southern California), with support from the EBRC Education Working Group. Audience These lecture slides are designed for educators looking to incorporate current synthetic & engineering biology practices into their teaching material. These slides were designed with a target audience of undergraduate and graduate students, but could be adapted for high school students (and coupled with BioBuilder material, for a great experience). Recommended student knowledge: “biology 101” level, generally how DNA & cells work Learning Objectives You will be able to answer: - What is synthetic/engineering biology? - How can I Design, Build, Test, and Learn from biological systems? - What are the Core Tools of engineering biology? - How and where can engineering biology be applied to positively impact society? You will have: Planned a design cycle to approach a current problem Learned about engineering biology tools that can help you develop your idea Discovered the many sectors that engineering biology can positively impact 

Abstract Flavodoxins (Flds) mediate the flux of electrons between oxidoreductases in diverse metabolic pathways. To investigate whether Flds can support electron transfer to a sulfite reductase (SIR) that evolved to couple with a ferredoxin, we evaluated the ability of Flds to transfer electrons from a ferredoxin‐NADP reductase (FNR) to a ferredoxin‐dependent SIR using growth complementation of anEscherichia colistrain with a sulfur metabolism defect. We show that Flds from cyanobacteria complement this growth defect when coexpressed with an FNR and an SIR that evolved to couple with a plant ferredoxin. When we evaluated the effect of peptide insertion on Fld‐mediated electron transfer, we observed a sensitivity to insertions within regions predicted to be proximal to the cofactor and partner binding sites, while a high insertion tolerance was detected within loops distal from the cofactor and within regions of helices and sheets that are proximal to those loops. Bioinformatic analysis showed that natural Fld sequence variability predicts a large fraction of the motifs that tolerate insertion of the octapeptide SGRPGSLS. These results represent the first evidence that Flds can support electron transfer to assimilatory SIRs, and they suggest that the pattern of insertion tolerance is influenced by interactions with oxidoreductase partners.