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These results demonstrates that a new state-of-the-art is available for making gene deletions in this promising bug. With a turnaround time that is 50% shorter than the current systems available, we believe that SIBR-Cas9 and SIBR2.0-Cas12a will help using C. necator in new P2X applications! 8/n

SIBR2.0 was then functional and we moved testing it for Cas12a. The new architectures revealed to be tight, allowing precise and controllable induction of cas12a as well, with similar efficiencies to the ones observed for Cas9! 7/n

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Here is where SIBR2.0 comes into place. Simona and Costas developed a script for strategically moving the intron position within the CDS to get rid of such hidden translation initiation starts. This was tested first in E. coli using GFP as readout. 6/n

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Then, we moved at testing if SIBR would work also on Cas12a. To our surprise, induction during the targeting assay revealed a leaky cas12a expression despite the presence of the intron with the STOP codon. This was because of a hidden translation start site that required some adjustments. 5/n

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We started by testing the SIBR setup on Cas9. This was because we already had evidence that it worked before, although poorly www.sciencedirect.com/science/arti... Targeting worked fine & by combining SIBR w/ Cas9, we achieved high editing efficiencies of >80% in two loci! Great start! 4/n

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Last, we assessed if we could sense glycolate for environmental monitoring purposes. We simulated phytoplankton blooms in the lab 🌞🌊(known to release glycolate) and used the sensors to detect any glycolate in the medium. We confirmed w/ HPLC that sensitive sensors worked for this purpose! 7/

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Now, moving to applications🏭. The most obvious one is in vivo enzyme screening. We did so by testing a small library of Mtk and Mcl (involved in CO2 fixating and carbon-conserving pathways) for testing their best expression levels. We identified the optimal RBS combination to support carbon flux 6/

Then, the masterpiece from @helenasm.bsky.social who characterized these strains under incremental concentrations of glyoxylate🦠. Overall, they ranged from uM to mM spanning 3 orders of magnitude🔍! We also left the possibility for the strains to sense glycolate, which was also confirmed 5/

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We deleted these genes in E. coli strains to convert them into metabolic sensors (few more KOs were needed). Great work here by Charlie, Ari, and Helena (and co) who did a great quality experimental job w/ impressive results. First, we confirmed the tightness of these deletions using 13C labeling 4/

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Here, Elad and Hai applied their recently developed medium-scale model to identify a set of KOs that can lead to auxotrophies for glyoxylate, a central metabolic hub around which is not trivial to design growth-coupled selection schemes. They identify 5/6 designs w/ increasing glyoxylate demand 😎 3/

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We just #published an end-to-end work for the computer-aided design💻, construction🛠️, validation✅, characterization🔬 & application💪 of #auxotroph metabolic sensors, the work-horses of in vivo growth-coupled selection screening systems-using glycolate as test case. 🧵👇1/ www.nature.com/articles/s41...

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To implement these solutions, we can use #auxotrophic metabolic sensors, coupling growth to target metabolic module activity. In this #preprint co-led with Helena Schulz, we develop a suite of glyoxylate and glycolate sensors with wide-range sensitivity & different scopes. 🔬🌱 doi.org/10.1101/2024...

In #biomanufacturing, controlling the #redox circuit is key to maximizing substrate-to-product conversion. In this opinion piece co-led with Javi and Maaike, we propose that orthogonal redox circuits can be a valuable tool for optimizing C1-biomanufacturing doi.org/10.1016/j.co...

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I work with non-model (autotrophic) #bacteria to improve them as #bioproduction hosts. In this manuscript co-led with Simona Della Valle, we upgraded the CRISPR/Cas toolkit of the C1 platform C. necator, achieving unprecedented efficiencies and turnover times. 🧬🔬 www.biorxiv.org/content/10.1...

Enrico Orsi @eorsi.bsky.social · 19/12/2024

As 2024 wraps up🌇, I’ve been reflecting on the journey in #metabolic #engineering this year. I’m grateful for the opportunity to contribute to cool research, and thought I’d share a thread of what I’ve been involved with. Looking forward to connecting with the #biotech community here on Bluesky! 🙏🔬

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To implement these solutions, we can create auxotrophic metabolic sensors, coupling growth to target metabolic module activity. In this #preprint co-led with Helena Schulz, we develop a suite of glyoxylate and glycolate sensors with wide-range sensitivity & different scopes doi.org/10.1016/j.co...

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Enrico Orsi @eorsi.bsky.social · 19/12/2024

In biomanufacturing, controlling the #redox circuit is key to maximizing substrate-to-product conversion. In this opinion piece co-led w/ Javi and Maaike, we propose that orthogonal redox circuits can be a valuable resource for optimizing C1-biomanufacturing. doi.org/10.1016/j.co...

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Enrico Orsi @eorsi.bsky.social · 19/12/2024