Improvement of the reverse tetracycline transactivator by single amino acid substitutions that reduce leaky target gene expression to undetectable levels

Roney, Ian J.; Rudner, Adam D.; Couture, Jean-François; Kærn, Mads

doi:10.1038/srep27697

Cited by 67 publications

(87 citation statements)

References 30 publications

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“…Regarding the system of doxycycline-inducible I-SceI expression, a variant of rtTA (rtTA-SEG72P) was used with a less leaky PTETO4 promoter 40 . The yeast strain used to amplify the rtTA variant and PTETO4 were provided by Mads Kaern.…”

Section: Construction Of Plasmids and Yeast Strainsmentioning

confidence: 99%

Quantitative insights into age-associated DNA-repair inefficiency in single cells

Young

Liu

Açar

2019

Preprint

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The double strand break (DSB) is a highly toxic form of DNA damage that is thought to be both a driver and consequence of age-related dysfunction. Although DSB repair is essential for a cell's survival, little is known about how DSB repair mechanisms are affected by cellular age. Here we characterize the impact of cellular aging on the efficiency of single-strand annealing (SSA), a repair mechanism for DSBs occurring between direct repeats. Using a single-cell reporter of SSA repair, we measure SSA repair efficiency in young and old cells, and report a 23.4% decline in repair efficiency. This decline is not due to increased usage of non-homologous end joining (NHEJ). Instead, we identify increased G1-phase duration in old cells as a factor responsible for the decreased SSA repair efficiency. We further explore how SSA repair efficiency is affected by sequence heterology and find that heteroduplex rejection remains high in old cells. Our work provides novel quantitative insights into the links between cellular aging and DSB repair efficiency at single-cell resolution in replicatively aging cells.

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Section: Construction Of Plasmids and Yeast Strainsmentioning

confidence: 99%

Quantitative insights into age-associated DNA-repair inefficiency in single cells

Young

Liu

Açar

2019

Preprint

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“…To generate a defined Msp1 substrate population prior to initiation of Msp1 activity, we devised a synthetic gene system for orthogonal drug-controlled expression of Pex15 and Msp1. Briefly, we created a yeast strain genetic background with two transcriptional activator-promoter pairs: 1. the doxycycline (DOX)-activated reverse tetracycline trans-activator (rTA) (Roney et al, 2016) Figure 1A and see below). This was followed by 2 hours of DOX wash-out to allow for mitochondrial maturation of newly-synthesized YFP-Pex15 ( Figure 1A).…”

Section: Inductionmentioning

confidence: 99%

“…For integration of the Z4EV transcription factor gene-ZD promoter cassette (McIsaac et al, 2013) upstream of MSP1, a PCR product containing a LEU marker, the Z4EV transcription factor cassette, and a GAL1 promoter variant altered for control by Z4EV was integrated immediately 5' to the MSP1 coding sequence. For doxycycline-induced expression of PEX15 variants, a similar PCR product was generated consisting of a TRP marker cassette, the G72V variant of the reverse tetracycline transactivator (rTA) (Roney et al, 2016), a GAL1 promoter variant altered for control by rTA, the YFP gene with no stop codon, the PEX15 gene or mutant variant, and the PEX15 terminator. This cassette was integrated into the strain's URA3 locus.…”

Section: Yeast Strain Constructionmentioning

confidence: 99%

The AAA Protein Msp1 Mediates Clearance of Excess Tail-Anchored Proteins from the Peroxisomal Membrane

Weir

Kamber

Martenson

et al. 2017

Preprint

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SUMMARYMsp1 is a conserved AAA ATPase in budding yeast localized to mitochondria where it prevents accumulation of mistargeted tail-anchored (TA) proteins, including the peroxisomal TA protein Pex15. Msp1 also resides on peroxisomes but it remains unknown how native TA proteins on mitochondria and peroxisomes evade Msp1 surveillance. We used live-cell quantitative cell microscopy tools and drug-inducible gene expression to dissect Msp1 function. We found that a small fraction of peroxisomal Pex15, exaggerated by overexpression, is turned over by Msp1.Kinetic measurements guided by theoretical modeling revealed that Pex15 molecules at mitochondria display age-independent Msp1 sensitivity. By contrast, Pex15 molecules at peroxisomes are rapidly converted from an initial Msp1-sensitive to an Msp1-resistant state. Lastly, we show that Pex15 interacts with the peroxisomal membrane protein Pex3, which shields Pex15 from Msp1-dependent turnover. In sum, our work argues that Msp1 selects its substrates on the basis of their solitary membrane existence.

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“…3C). Notably, different from other aTF engineering efforts 43 , the increase in dynamic range was largely due to increased expression upon ligand induction, as only modest changes in OFF state expression was observed for the dynamic range variants compared to WT BenM (Fig. 2A).…”

Section: Resultsmentioning

confidence: 67%

Evolution-guided engineering of small-molecule biosensors

Snoek

Chaberski

Ambri

et al. 2019

Preprint

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19Allosteric transcription factors (aTFs) have proven widely applicable for 20 biotechnology and synthetic biology as ligand-specific biosensors enabling real-time 21 monitoring, selection and regulation of cellular metabolism. However, both the 22 biosensor specificity and the correlation between ligand concentration and biosensor 23 output signal, also known as the transfer function, often needs to be optimized before 24 meeting application needs. Here, we present a versatile and high-throughput method 25 to evolve and functionalize prokaryotic aTF specificity and transfer functions in a 26 eukaryote chassis, namely baker's yeast Saccharomyces cerevisiae. From a single 27 round of directed evolution of the effector-binding domain (EBD) coupled with various 28 2 toggled selection regimes, we robustly select aTF variants of the cis, cis-muconic 29 acid-inducible transcription factor BenM evolved for change in ligand specificity, 30 increased dynamic output range, shifts in operational range, and a complete 31 inversion of function from activation to repression. Importantly, by targeting only the 32 EBD, the evolved biosensors display DNA-binding affinities similar to BenM, and are 33 functional when ported back into a non-native prokaryote chassis. The developed 34 platform technology thus leverages aTF evolvability for the development of new host-35 agnostic biosensors with user-defined small-molecule specificities and transfer 36 functions. 37 38 39 40 48 mammalian cell differentiation, and synthetic cell-cell communication devices 2-5 . 49 Given the large number of allosteric transcription factors (aTFs) present in the 50 prokaryotic kingdom 6 , the diversity of chemical structures recognized 7 , and their 51 modular domain structure encoded by a conserved DNA-binding domain (DBD) 52 linked to a diversified effector-binding domain (EBD) 8 , small-molecule biosensors 53 based on aTFs are a particularly valuable class of genetic switches. Ongoing 54biosensor research therefore seeks to prospect new biosensors from genomic 55 resources 9,10 , while also developing general design rules and engineering strategies 56 3 from existing aTFs. Indeed, due to the modular structure of aTFs, several studies 57 have successfully adopted EBD-swapping strategies into platform DBDs to rationally 58 engineer new aTF logic 11-13 , while engineering EBD destabilization for ligand-59 controlled biosensor stability also have proven successful [14][15][16] . However, these 60 rational design strategies for engineering new biosensors suffer from the introduction 61 of cross-talk between ligand specificities, difficulty in creating chimeras from different 62 aTF superfamilies, and the risk of losing allostery 2,11,17 . Ultimately, this may impact 63 several aspects of biosensor performance, such as the operational and dynamic 64 output ranges, the specificity, and mode-of-action -collectively referred to as the 65 biosensor transfer function or logic. 66 Acknowledging that allosteric regulation relies on complex interdom...

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Improvement of the reverse tetracycline transactivator by single amino acid substitutions that reduce leaky target gene expression to undetectable levels

Cited by 67 publications

References 30 publications

Quantitative insights into age-associated DNA-repair inefficiency in single cells

Quantitative insights into age-associated DNA-repair inefficiency in single cells

The AAA Protein Msp1 Mediates Clearance of Excess Tail-Anchored Proteins from the Peroxisomal Membrane

Evolution-guided engineering of small-molecule biosensors

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