Bacterial Microcolonies in Gel Beads for High-Throughput Screening of Libraries in Synthetic Biology

Duarte, José M.; Barbier, Içvara; Schaerli, Yolanda

doi:10.1021/acssynbio.7b00111

Cited by 45 publications

(47 citation statements)

References 40 publications

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“…Instead, therefore, we switched to growing reporter cells inside alginate beads to microcolonies, which improved the reproducibility and screening efficiency, and reduced the number of false positives. Others have recently compared such procedures and have come to the same conclusion 31 . Surprisingly, around 0.5 % of the clones in our library were constitutively 'ON', showing high GFP fluorescence even in absence of inducer.…”

Section: Discussionmentioning

confidence: 78%

Computational redesign of theEscherichia coliribose-binding protein ligand binding pocket for 1,3-cyclohexanediol and cyclohexanol

Tavares

Reimer

Roy

et al. 2019

Preprint

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Tel: +41 21 692 5630. 14 15 Running title: Ligand binding pocket redesign of Ribose-binding protein 16 Keywords: Rosetta, RbsB, ribose, 1,3-cyclohexanediol, reporter bacteria, fluorescence-assisted bead 17 sorting, protein folding 18 19 20 2 21 Bacterial periplasmic-binding proteins have been acclaimed as general biosensing platform, but 22 their range of natural ligands is too limited for optimal development of chemical compound 23 detection. Computational redesign of the ligand-binding pocket of periplasmic-binding proteins 24 may yield variants with new properties, but, despite earlier claims, genuine changes of specificity 25 to non-natural ligands have so far not been achieved. In order to better understand the reasons of 26 such limited success, we revisited here the Escherichia coli RbsB ribose-binding protein, aiming to 27 achieve perceptible transition from ribose to structurally related chemical ligands 1,3-28 cyclohexanediol and cyclohexanol. Combinations of mutations were computationally predicted for 29 nine residues in the RbsB binding pocket, then synthesized and tested in an E. coli reporter chassis. 30 Two million variants were screened in a microcolony-in-bead fluorescence-assisted sorting 31 procedure, which yielded six mutants no longer responsive to ribose but with 1.2-1.5 times 32 induction in presence of 1 mM 1,3-cyclohexanediol, one of which responded to cyclohexanol as 33 well. Isothermal microcalorimetry confirmed 1,3-cyclohexanediol binding, although only two 34 mutant proteins were sufficiently stable upon purification. Circular dichroism spectroscopy 35 indicated discernable structural differences between these two mutant proteins and wild-type 36 RbsB. This and further quantification of periplasmic-space abundance suggested most mutants to 37 be prone to misfolding and/or with defects in translocation compared to wild-type. Our results 38 thus affirm that computational design and library screening can yield RbsB mutants with 39 recognition of non-natural but structurally similar ligands. The inherent arisal of protein instability 40 or misfolding concomitant with designed altered ligand-binding pockets should be overcome by 41 new experimental strategies or by improved future protein design algorithms. 42 43 Periplasmic binding proteins (PBPs) form a versatile superfamily of proteins with a conserved protein 44 structure, named the bilobal structural fold 1,2 . PBPs facilitate nutrient and trace mineral scavenging 45 for bacterial cells, by binding the ligand in the periplasmic space at high affinity and delivering the 46 bound-ligand to a specific membrane-spanning transport channel 2 . Some PBPs are additionally 47 involved in chemotactic sensing and interact in the ligand-bound state with a membrane-located 48 chemoreceptor 3 . The crystal structures of several PBPs have been determined, showing two domains 49 connected by a hinge region, with the binding pocket located between the two domains 3 . Both 50 structure and nuclear-magnetic resonance data indicate that PBPs switch betw...

show abstract

Section: Discussionmentioning

confidence: 78%

Computational redesign of theEscherichia coliribose-binding protein ligand binding pocket for 1,3-cyclohexanediol and cyclohexanol

Tavares

Reimer

Roy

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…The potential for combinatorial assembly is a desirable feature of DNA assembly methods, since it enables the building of large numbers of constructs without the need to design and build them individually. These libraries can then be rapidly tested provided that an appropriate high-throughput screening method is put in place 25,26 .…”

Section: Resultsmentioning

confidence: 99%

A Framework for the Modular and Combinatorial Assembly of Synthetic Gene Circuits

Santos‐Moreno

Schaerli

2019

ACS Synth. Biol.

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Synthetic gene circuits emerge from iterative design-build-test cycles. Most commonly, the time-limiting step is the circuit construction process. Here, we present a hierarchical cloning scheme based on the widespread Gibson assembly method and make the set of constructed plasmids freely available. Our two-step modular cloning scheme allows for simple, fast, efficient and accurate assembly of gene circuits and combinatorial circuit libraries in Escherichia coli. The first step involves Gibson assembly of transcriptional units from constituent parts into individual intermediate plasmids. In the second step, these plasmids are digested with specific sets of restriction enzymes. The resulting flanking regions have overlaps that drive a second Gibson assembly into a single plasmid to yield the final circuit. This approach substantially reduces time and sequencing costs associated with gene circuit construction and allows for modular and combinatorial assembly of circuits. We demonstrate the usefulness of our framework by assembling a CRISPR-based double-inverter circuit and a combinatorial library of 3-node networks.

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“…To apply this system in methanotroph screening in the future, the procedure of phenotyping and sorting of microbes of interest needs to be further improved. A gelling agent could be incorporated into our flexible assay system for imaging and sorting of GESS-methanotroph compartments by flow cytometry in a high-throughput manner (Duarte et al, 2017). Addition of gelling reagents would make the GESSlet a powerful system for screening desired methanotrophs for developing industrially applicable enzymes and engineering microbial cell factories based on cell-to-cell communication.…”

Section: Functional Phenotyping Of Methanotrophs With Smmo Activitymentioning

confidence: 99%

Sensitive and Rapid Phenotyping of Microbes With Soluble Methane Monooxygenase Using a Droplet-Based Assay

Lee

Baek

Kim

et al. 2020

Front. Bioeng. Biotechnol.

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Methanotrophs with soluble methane monooxygenase (sMMO) show high potential for various ecological and biotechnological applications. Here, we developed a high throughput method to identify sMMO-producing microbes by integrating droplet microfluidics and a genetic circuit-based biosensor system. sMMO-producers and sensor cells were encapsulated in monodispersed droplets with benzene as the substrate and incubated for 5 h. The sensor cells were analyzed as the reporter for phenol-sensitive transcription activation of fluorescence. Various combinations of methanotrophs and biosensor cells were investigated to optimize the performance of our droplet-integrated transcriptional factor biosensor system. As a result, the conditions to ensure sMMO activity to convert the starting material, benzene, into phenol, were determined. The biosensor signals were sensitive and quantitative under optimal conditions, showing that phenol is metabolically stable within both cell species and accumulates in picoliter-sized droplets, and the biosensor cells are healthy enough to respond quantitatively to the phenol produced. These results show that our system would be useful for rapid evaluation of phenotypes of methanotrophs showing sMMO activity, while minimizing the necessity of time-consuming cultivation and enzyme preparation, which are required for conventional analysis of sMMO activity.

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Bacterial Microcolonies in Gel Beads for High-Throughput Screening of Libraries in Synthetic Biology

Cited by 45 publications

References 40 publications

Computational redesign of theEscherichia coliribose-binding protein ligand binding pocket for 1,3-cyclohexanediol and cyclohexanol

Computational redesign of theEscherichia coliribose-binding protein ligand binding pocket for 1,3-cyclohexanediol and cyclohexanol

A Framework for the Modular and Combinatorial Assembly of Synthetic Gene Circuits

Sensitive and Rapid Phenotyping of Microbes With Soluble Methane Monooxygenase Using a Droplet-Based Assay

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