The Laboratory Automation Protocol (LAP) Format and Repository: A Platform for Enhancing Workflow Efficiency in Synthetic Biology

Anhel, Ana-Mariya; Alejaldre, Lorea; Goñi-Moreno, Ángel

doi:10.1021/acssynbio.3c00397

Cited by 4 publications

(1 citation statement)

References 33 publications

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“…Once pTn7-M, pBAMD1.2 and pBLAM1.2 derivative suicide plasmids were built, they were delivered into P. putida KT2440 chromosome following a conjugation protocol previously described for transpositions into gram-negative bacteria [50] [51]). Conjugation outcome was processed in order to separate variants, discard spurious integrations and genotype the transposon location in accordance with an automated high-throughput screening method [63] using LAP protocols [64]. Selected and genotyped libraries were stored at -80ºC for further phenotypic characterization.…”

Section: Construction Selection and Genotyping Of Transposon Insertio...mentioning

confidence: 99%

Reprogramming genetic circuits using space

Alejaldre,

Miró-Bueno,

Hueso-Gil

et al. 2024

Preprint

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Genetic circuits confer computing abilities to living cells, performing novel transformations of input stimuli into output responses. These genetic circuits are routinely engineered for insertion into bacterial plasmids and chromosomes, using a design paradigm whose only spatial consideration is a linear ordering of the individual components. However, chromosomal DNA has a complex three dimensional conformation which alters the mechanics of gene expression, leading to dynamics that are specific to chromosomal location. Here we demonstrate that because of this, position in the bacterial chromosome is crucial to the function of synthetic genetic circuits, and that three dimensional space should not be overlooked in their design. Our results show that genetically identical circuits can be reprogrammed to produce different outputs by changing their spatial positioning and configuration. We engineer 221 spatially unique genetic circuits of four different types, three regulatory cascades and a toggle switch, by either inserting the entire circuit in a specific chromosomal position or separating and distributing circuit modules. Their analysis reveals that spatial positioning can be used not only to optimize circuits but also to switch circuits between modes of operation, giving rise to new functions. Alongside a comprehensive characterization of chromosomal space using single-cell RNA-seq profiles and Hi-C interaction maps, we offer baseline information for leveraging intracellular space as a design parameter in bioengineering.

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Section: Construction Selection and Genotyping Of Transposon Insertio...mentioning

confidence: 99%

Reprogramming genetic circuits using space

Alejaldre,

Miró-Bueno,

Hueso-Gil

et al. 2024

Preprint

Self Cite

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Workflow to Select Functional Promoter DNA Baits and Screen Arrayed Gene Libraries in Yeast

Fañanás‐Pueyo,

Anhel,

Goñi‐Moreno

et al. 2024

Current Protocols

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The yeast one‐hybrid system (Y1H) is used extensively to identify DNA–protein interactions. The generation of large collections of open reading frames (ORFs) to be used as prey in screenings is not a bottleneck nowadays and can be carried out in‐house or offered as a service by companies. However, the straightforward use of full gene promoters as baits to identify interacting proteins undermines the accuracy and sensitivity of the assay, especially in the case of multicellular eukaryotes. Therefore, it is paramount to implement procedures for efficient identification of suitable promoter fragments compatible with the Y1H assay. Here, we describe a workflow to identify biologically relevant conserved promoter fragments of Arabidopsis thaliana through simple and robust phylogenetic analyses. Additionally, we describe a manual method and its automated robotized version for rapid and efficient high‐throughput Y1H screenings of arrayed ORF libraries with the identified DNA fragments. Moreover, this method can be scaled up or down and used for yeast two‐hybrid screenings to search for possible interactors of proteins identified by the Y1H approach or any other protein of interest, altogether underscoring its suitability to build gene regulatory networks. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Selection of DNA baits for Y1H screeningsBasic Protocol 2: Y1H screenings with arrayed gene librariesAlternate Protocol: Automated screening with a liquid‐handling robot

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