The restriction free (RF) cloning has emerged as one of the highly efficient techniques in the area of genetic engineering. RF cloning has wide range of applications in plasmid DNA manipulation including cloning of a single gene, simultaneous assembly of multiple DNA fragments, and mutagenesis from single to multiple simultaneous alterations of a target DNA. Recently, we have developed a new technique of circular permutation using RF cloning. Circular permutation is widely used to investigate the mechanisms of protein folding and function. Previously, restriction enzyme based cloning was used to introduce circular permutation. Our RF cloning method made the protocol faster and more cost-effective. In this chapter, we describe a step-by-step protocol for generating circular permutants using RF methodology.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1007/978-1-0716-2152-3_10 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Backbone resonance assignments of PhoCl, a photocleavable protein.
Biomol NMR Assign
January 2025
High Magnetic Field Laboratory, Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui, China.
PhoCl is a photocleavable protein engineered from a green-to-red photoconvertible fluorescent protein by circular permutation, and has been used in various optogenetic applications including precise control of protein localization and activity in cells. Upon violet light illumination, PhoCl undergoes a β-elimination reaction to be cleaved at the chromophore, resulting in spontaneous dissociation into a large empty barrel and a small C-terminal peptide. However, the structural determinants and the mechanism of the PhoCl photocleavage remain elusive, hindering the further development of more robust photocleavable optogenetic tools.
View Article and Find Full Text PDFSystematic characterization of indel variants using a yeast-based protein folding sensor.
Structure
December 2024
Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark. Electronic address:
Gene variants resulting in insertions or deletions of amino acid residues (indels) have important consequences for evolution and are often linked to disease, yet, compared to missense variants, the effects of indels are poorly understood and predicted. We developed a sensitive protein folding sensor based on the complementation of uracil auxotrophy in yeast by circular permutated orotate phosphoribosyltransferase (CPOP). The sensor reports on the folding of disease-linked missense variants and de-novo-designed proteins.
View Article and Find Full Text PDFExplainable Artificial Intelligence to Predict the Water Status of Cotton ( L., 1763) from Sentinel-2 Images in the Mediterranean Area.
Plants (Basel)
November 2024
Council for Agricultural Research and Economics, Research Center for Agriculture and Environment, Via Celso Ulpiani, 5, 70125 Bari, Italy.
Climate change and water scarcity bring significant challenges to agricultural systems in the Mediterranean region. Novel methods are required to rapidly monitor the water stress of the crop to avoid qualitative losses of agricultural products. This study aimed to predict the stem water potential of cotton ( L.
View Article and Find Full Text PDFEfficient circular RNA synthesis for potent rolling circle translation.
Nat Biomed Eng
December 2024
MRC Laboratory of Molecular Biology, Cambridge, UK.
Circular RNA (circRNA) is a candidate for next-generation messenger RNA therapeutics owing to its remarkable stability. Here we describe trans-splicing-based methods for the synthesis of circRNAs over 8,000 nucleotides. The methods are independent of bacterial sequences, outperform the permuted intron-exon method and allow for the incorporation of RNA modifications.
View Article and Find Full Text PDFCHiTA: A scarless high-throughput pipeline for characterization of ribozymes.
Methods
December 2024
Department of Chemistry, Pennsylvania State University, University Park, PA 16802, USA; Center for RNA and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA; Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA. Electronic address:
Small self-cleaving ribozymes are catalytic RNAs that cleave their phosphodiester backbone rapidly and site-specifically, without the assistance of proteins. Their catalytic properties make them ideal targets for applications in RNA pharmaceuticals and bioengineering. Consequently, computational pipelines that predict or design thousands of self-cleaving ribozyme candidates have been developed.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!