Split intein-mediated selection of cells containing two plasmids using a single antibiotic

Palanisamy, Navaneethan; Degen, Anna; Morath, Anna; Ballestin, Jara Ballestin; Juraske, Claudia; Öztürk, Mehmet Ali; Sprenger, Georg A.; Youn, Jung-Won; Schamel, Wolfgang W. A.; Ventura, Barbara Di

doi:10.1038/s41467-019-12911-1

Cited by 24 publications

(54 citation statements)

References 47 publications

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“…It has a resolved crystal structure and a well-established split site at 194/196 36 or 195/196 37 . The previously mentioned computational method identified a new split site at 104/105, which when used with the split gp41-1 intein, allowed co-selection of two plasmids with just ampicillin 25 . We therefore sought to recover these two sites with IBM.…”

Section: Resultsmentioning

confidence: 99%

“…The computational method by Palanisamy et al 25 , and the SPELL algorithm 26 , both mentioned above, suggest split sites based on protein 3D structures and works in silico, whereas our IBM/acVHH-assisted BM workflow takes on an empirical approach and addresses the same issue with different constraints. Sometimes solved 3D structures may be unavailable and de novo structure prediction might not sufficiently reflect the multimerization required in some proteins like TetR.…”

Section: Discussionmentioning

confidence: 99%

“…Since a computational method 25 exists to predict split sites for the gp41-1 intein 35 on antibiotic resistance genes, we asked how a library approach through IBM would perform in comparison to a computational approach. We focused on the case of TEM-1 β-lactamase (also abbreviated as BLA in figures).…”

Section: Ibm Identified a Computationally Unpredicted Split Site On βmentioning

confidence: 99%

“…A better solution would be to predict split sites computationally from protein crystal structures. A method was recently developed to predict intein-insertable split sites by searching flexible regions on protein structures and regions that lack functional conservation 25 . This was demonstrated on antibiotic resistance genes.…”

Section: Introductionmentioning

confidence: 99%

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Intein-assisted bisection mapping systematically splits proteins for Boolean logic and inducibility engineering

Tyh

Shao

et al. 2020

Preprint

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Split inteins are powerful tools for seamless ligation of synthetic split proteins. Yet, their use remains limited because the already intricate split site identification problem is often complicated by the requirement of extein junction sequences. To address this, we augmented a mini-Mu transposon-based screening approach and devised the intein-assisted bisection mapping (IBM) method. IBM robustly revealed clusters of split sites on five proteins, converting them into AND or NAND logic gates. We further showed that the use of inteins expands functional sequence space for splitting a protein. We also demonstrated the utility of our approach over rational inference of split sites from secondary structure alignment of homologous proteins. Furthermore, the intein inserted at an identified site could be engineered by the transposon again to become partially chemically inducible, and to some extent enabled post-translational tuning on host protein function. Our work offers a generalizable and systematic route towards creating split protein-intein fusions and conditional inteins for protein activity control.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Ibm Identified a Computationally Unpredicted Split Site On βmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Intein-assisted bisection mapping systematically splits proteins for Boolean logic and inducibility engineering

Tyh

Shao

et al. 2020

Preprint

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“…We have recently devised a method based on s plit i ntein- m ediated enzyme reconstitution, that allows selecting bacterial and mammalian cells containing two p lasmids with a single antibiotic, which we named SiMPl 1 . Inteins are proteins that excise themselves out of host proteins in an autocatalytic reaction that results with a peptide bond joining the polypeptides originally flanking the intein 2, 3 .…”

mentioning

confidence: 99%

Expanding the SiMPl plasmid toolbox for use with spectinomycin/streptomycin

Palanisamy¹,

Ballestin²,

Ventura³

2021

Preprint

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We recently developed the SiMPl plasmid toolbox, which is constituted by pairs of plasmids, generically indicated as pSiMPlx_N and pSiMPlx_C, which can be stably maintained in Escherichia coli with a single antibiotic x. The method exploits the split intein gp41-1 to reconstitute the enzyme conferring resistance towards the antibiotic x, whereby each enzyme fragment is expressed from one of the plasmids in the pair. pSiMPl plasmids are currently available for use with ampicillin, kanamycin, chloramphenicol, hygromycin and puromycin. Here we introduce another pair for use with spectinomycin/streptomycin broadening the application spectrum of the SiMPl toolbox. To find functional splice sites in aminoglycoside adenylyltransferase we apply a streamlined strategy looking exclusively at the flexibility of native cysteine and serine residues, which we first validated splitting the enzymes conferring resistance towards ampicillin, kanamycin, chloramphenicol and hygromycin. This strategy could be used in the future to split other enzymes conferring resistance towards antibiotics.

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Real‐Time, Non‐Invasive Monitoring of Neuronal Differentiation Using Intein‐Enabled Fluorescence Signal Translocation in Genetically Encoded Stem Cell‐Based Biosensors

Lee,

Choi,

Kwon

et al. 2024

Adv Funct Materials

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Real‐time and non‐invasive monitoring of neuronal differentiation helps to increase understanding of neuronal development and develop stem cell therapies for neurodegenerative diseases. Conventional methods such as RT‐PCR, western blotting, and immunofluorescence (IF), lack single‐cell‐level resolution and require invasive procedures, fixation, and staining. These limitations hinder accurate monitoring progress of neural stem cell (NSC) differentiation and understanding its functions. Herein, a novel approach is reported to non‐invasively monitor neuronal differentiation in real‐time using cell‐based biosensors (CBBs) that detects hippocalcin, biomarker of neuronal differentiation. To construct the hippocalcin sensor proteins, two different hippocalcin bioreceptors are fused to each split‐intein, carrying split‐nuclear localization signal (NLS) peptides, respectively, and fluorescent protein is introduced as reporter. CBBs operated in the presence of hippocalcin to generate functional signal peptides, which promptly translocated the fluorescence signal to the nucleus. The NSC‐based biosensor shows fluorescence signal translocation only upon neuronal differentiation and not undifferentiated stem cells or glial cells. Furthermore, this approach allows monitoring of neural differentiation at earlier stages than detected using IF staining. It is believed that novel CBBs offer an alternative to current techniques by capturing the dynamics of differentiation progress at the single‐cell‐level and providing a tool to evaluate how NSCs efficiently differentiate into neurons.

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Split intein-mediated selection of cells containing two plasmids using a single antibiotic

Cited by 24 publications

References 47 publications

Intein-assisted bisection mapping systematically splits proteins for Boolean logic and inducibility engineering

Intein-assisted bisection mapping systematically splits proteins for Boolean logic and inducibility engineering

Expanding the SiMPl plasmid toolbox for use with spectinomycin/streptomycin

Real‐Time, Non‐Invasive Monitoring of Neuronal Differentiation Using Intein‐Enabled Fluorescence Signal Translocation in Genetically Encoded Stem Cell‐Based Biosensors

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