Engineering orthogonal dual transcription factors for multi-input synthetic promoters

Brödel, Andreas K.; Jaramillo, Alfonso; Isalan, Mark

doi:10.1038/ncomms13858

Cited by 55 publications

(94 citation statements)

References 44 publications

(69 reference statements)

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“…The method has been used to evolve a set of dual activator-repressor switches for orthogonal logic gates, based on bacteriophage λ cI variants, and multi-input promoter architectures, and these switches have been successfully applied in downstream synthetic gene circuits 19 . In general, the method is capable of evolving any gene-or gene circuit function-on the PM that can be linked to pVI production.…”

Section: Applications Of the Methodsmentioning

confidence: 99%

“…The selection process takes place inside E. coli cells by linking the target protein's activity to conditional phage production, thus allowing enrichment of functional library members. This is exemplified here by the directed evolution of orthogonal dual TFs based on bacteriophage λ cI variants 19 , selecting against synthetic promoters. However, the method can be readily adapted for other target biomolecules ("Applications of the method").…”

Section: Overview Of the Protocolmentioning

confidence: 99%

“…The ratio of green to red colonies should increase over time because the nonactive TF cI 5G6G,P results in red colonies, whereas the enriched active cI opt leads to green colonies because of GFP activation and mCherry repression. As an alternative control selection, one can replace the TF cI 5G6G,P with a reporter (e.g., a red fluorescent protein (RFP)) on the PM and then monitor the selection process by infecting TG1 cells and counting the ratio of red to white colonies after each round of selection 19 . …”

Section: Experimental Designmentioning

confidence: 99%

“…In vivo methods promote selection for orthogonality 17,18 -a lack of cross-reactions-by intrinsically counterselecting against adverse effects inside the cell. To further broaden the applications of in vivo directed evolution, we recently developed an M13-phage-based method 19 for the intracellular selection of proteins from combinatorial libraries with distinct differences and advantages to established techniques.…”

Section: Introductionmentioning

confidence: 99%

“…These mutations can originate either from mutations during library cloning or from the spontaneous error rate of M13 phage replication, which is ~0.0046 mutations per genome per replication 43 . Such mutations can provide function 19 and contribute to directed evolution.…”

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Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication

2017

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Directed evolution is a powerful tool to improve the characteristics of biomolecules. Here we present a protocol for the intracellular evolution of proteins with distinct differences and advantages in comparison with established techniques. these include the ability to select for a particular function from a library of protein variants inside cells, minimizing undesired coevolution and propagation of nonfunctional library members, as well as allowing positive and negative selection logics using basally active promoters. a typical evolution experiment comprises the following stages: (i) preparation of a combinatorial M13 phagemid (pM) library expressing variants of the gene of interest (GoI) and preparation of the Escherichia coli host cells; (ii) multiple rounds of an intracellular selection process toward a desired activity; and (iii) the characterization of the evolved target proteins. the system has been developed for the selection of new orthogonal transcription factors (tFs) but is capable of evolving any gene-or gene circuit function-that can be linked to conditional M13 phage replication. Here we demonstrate our approach using as an example the directed evolution of the bacteriophage l cI tF against two synthetic bidirectional promoters. the evolved tF variants enable simultaneous activation and repression against their engineered promoters and do not cross-react with the wild-type promoter, thus ensuring orthogonality. this protocol requires no special equipment, allowing synthetic biologists and general users to evolve improved biomolecules within ~7 weeks.© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. the chances of propagating nonfunctional library members because of multiple infections. A GOI with the desired characteristics upregulates gene VI expression on the AP, completing the phage life cycle. For example, a randomized TF library member that activates an artificial promoter upstream of gVI will increase its own phage production (Fig. 4a). In this way, a protein with novel desired properties can be selected after several rounds of reinfection. Applications of the methodThe method has been used to evolve a set of dual activator-repressor switches for orthogonal logic gates, based on bacteriophage λ cI variants, and multi-input promoter architectures, and these switches have been successfully applied in downstream synthetic gene circuits 19 . In general, the method is capable of evolving any gene-or gene circuit function-on the PM that can be linked to pVI production. This is analogous to previous uses of phageassisted continuous evolution (PACE) 22 (Fig. 4). With PACE, a wide range of medically and biotechnologically relevant biomolecules, including polymerases 22 , proteases 23 and genome-editing proteins 10 , as well as protein-protein interactions 24 , were linked to conditional M13 phage propagation. In principle, any application in which directed evolution approaches have been...

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Section: Applications Of the Methodsmentioning

confidence: 99%

Section: Overview Of the Protocolmentioning

confidence: 99%

Section: Experimental Designmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication

2017

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Continuous Evolution of ProteinsIn Vivo

Wellner

Ravikumar

Liu

2021

Protein Engineering

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Controlling Cells with Light and LOV

et al. 2018

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Optogenetics is a powerful method for studying dynamic processes in living cells and has advanced cell biology research over the recent past. Key to the successful application of optogenetics is the careful design of the light‐sensing module, typically employing a natural or engineered photoreceptor that links the exogenous light input to the cellular process under investigation. Light–oxygen–voltage (LOV) domains, a highly diverse class of small blue light sensors, have proven to be particularly versatile for engineering optogenetic input modules. These can function via diverse modalities, including inducible allostery, protein recruitment, dimerization, or dissociation. This study reviews recent advances in the development of LOV domain‐based optogenetic tools and their application for studying and controlling selected cellular functions. Focusing on the widely employed LOV2 domain from Avena sativa phototropin‐1, this review highlights the broad spectrum of engineering opportunities that can be explored to achieve customized optogenetic regulation. Finally, major bottlenecks in the development of optogenetic methods are discussed and strategies to overcome these with recent synthetic biology approaches are pointed out.

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Engineering orthogonal dual transcription factors for multi-input synthetic promoters

Cited by 55 publications

References 44 publications

Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication

Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication

Continuous Evolution of ProteinsIn Vivo

Controlling Cells with Light and LOV

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