The AssemblX Toolkit for Reliable and User-Friendly Multigene Assemblies

Machens, Fabian; Hochrein, Lena

doi:10.1007/978-1-0716-0908-8_3

Cited by 1 publication

(4 citation statements)

References 14 publications

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“…Multi-gene constructs were assembled using the AssemblX toolkit, , which enables level-based multi-part assemblies. While Level 0 plasmids are generally employed to assemble single transcriptional units (TUs), Level 1 vectors allow the combination of up to five Level 0 modules.…”

Section: Methodsmentioning

confidence: 99%

“…The plasmids with the different variations of the dCas9-based red light-inducible TFs (Figure A) were assembled using the AssemblX toolkit. , The final Level 1 constructs (given in Supporting Information Table S4) were built from preassembled Level 0 plasmids by in vivo recombination in yeast . Level 0 plasmids were assembled using NEBuilder HiFi DNA Assembly Master Mix.…”

Section: Methodsmentioning

confidence: 99%

“…To allow assembly of multiple sgRNAs into a single array, the multi-sgRNA expression plasmids pFM149-pFM153 were designed based on the previously published AssemblX vector series pL0_A0_1; pL0_B0_1; pL0_C0_1; pL0_D0_1; and pL0_E0_1. , To achieve compatibility with the Golden Gate strategy for sgRNA multimerization, we deleted the Bsm BI sites in these vectors. A ccdB selection cassette flanked by assembly connectors L1 and Rx was inserted between the AssemblX homology regions of each vector to yield plasmids pFM0149-pFM0153.…”

Section: Methodsmentioning

confidence: 99%

“…Level 0 constructs pLH_201-203 and Level 1 construct pLH_204 were cloned with the AssemblX toolkit. , Templates and primers for PCR amplification of Level 0 parts are given in Supporting Information Table S5. Level 0 plasmids pLH_201 and pLH_202 were used to assemble Level 1 construct pLH_204 (Supporting Information Table S4).…”

Section: Methodsmentioning

confidence: 99%

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PhiReX 2.0: A Programmable and Red Light-Regulated CRISPR-dCas9 System for the Activation of Endogenous Genes in Saccharomyces cerevisiae

et al. 2023

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Metabolic engineering approaches do not exclusively require fine-tuning of heterologous genes but oftentimes also modulation or even induction of host gene expression, e.g., in order to rewire metabolic fluxes. Here, we introduce the programmable red light switch PhiReX 2.0, which can rewire metabolic fluxes by targeting endogenous promoter sequences through single-guide RNAs (sgRNAs) and activate gene expression in Saccharomyces cerevisiae upon red light stimulation. The split transcription factor is built from the plant-derived optical dimer PhyB and PIF3, which is fused to a DNA-binding domain based on the catalytically dead Cas9 protein (dCas9) and a transactivation domain. This design combines at least two major advantages: first, the sgRNAs, guiding dCas9 to the promoter of interest, can be exchanged in an efficient and straightforward Golden Gate-based cloning approach, which allows for rational or randomized combination of up to four sgRNAs in a single expression array. Second, target gene expression can be rapidly upregulated by short red light pulses in a light dose-dependent manner and returned to the native expression level by applying far-red light without interfering with the cell culture. Using the native yeast gene CYC1 as an example, we demonstrated that PhiReX 2.0 can upregulate CYC1 gene expression by up to 6-fold in a light intensity-dependent and reversible manner using a single sgRNA.

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Section: Methodsmentioning

confidence: 99%