Editor
Noelia Foresi
  • Research Associate, Universidad Nacional de Mar del Plata
Research fields
  • Molecular Biology
CRISPR-Cas9 Protocol for Efficient Gene Knockout and Transgene-free Plant Generation
Authors:  Enzo A. Perk, Ana M. Laxalt and Ignacio Cerrudo, date: 06/05/2024, view: 1001, Q&A: 0

Gene editing technologies have revolutionized plant molecular biology, providing powerful tools for precise gene manipulation for understanding function and enhancing or modifying agronomically relevant traits. Among these technologies, the CRISPR-Cas9 system has emerged as a versatile and widely accepted strategy for targeted gene manipulation. This protocol provides detailed, step-by-step instructions for implementing CRISPR-Cas9 genome editing in tomato plants, with a specific focus in generating knockout lines for a target gene. For that, the guide RNA should preferentially be designed within the first exon downstream and closer to the start codon. The edited plants obtained are free of transgene cassette for expression of the CRISPR-Cas9 machinery.

Autolysin Production from Chlamydomonas reinhardtii
Author:  Justin Findinier, date: 07/05/2023, view: 518, Q&A: 1

Chlamydomonas reinhardtii is a model organism for various processes, from photosynthesis to cilia biogenesis, and a great chassis to learn more about biofuel production. This is due to the width of molecular tools available, which have recently expanded with the development of a modular cloning system but, most importantly, with CRISPR/Cas9 editing now being possible. This technique has proven to be more efficient in the absence of a cell wall by using specific mutants or by digesting Chlamydomonas cell wall using the mating-specific metalloprotease autolysin (also called gametolysin). Multiple protocols have been used and shared for autolysin production from Chlamydomonas cells; however, they provide very inconsistent results, which hinders the capacity to routinely perform CRISPR mutagenesis. Here, we propose a simple protocol for autolysin production requiring transfer of cells from plates into a dense liquid suspension, gametogenesis by overnight incubation before mixing of gametes, and enzyme harvesting after 2 h. This protocol has shown to be highly efficient for autolysin production regardless of precise control over cell density at any step. Requiring a minimal amount of labor, it will provide a simple, ready-to-go approach to produce an enzyme critical for the generation of targeted mutants.


Graphical overview



Workflow for autolysin production from Chlamydomonas reinhardtii

Site-specific Incorporation of Phosphoserine into Recombinant Proteins in Escherichia coli
Authors:  Phillip Zhu, Ryan A. Mehl and Richard B. Cooley, date: 11/05/2022, view: 1986, Q&A: 0

This protocol describes the recombinant expression of proteins in E. coli containing phosphoserine (pSer) installed at positions guided by TAG codons. The E. coli strains that can be used here are engineered with a ∆serB genomic knockout to produce pSer internally at high levels, so no exogenously added pSer is required, and the addition of pSer to the media will not affect expression yields. For “truncation-free” expression and improved yields with high flexibility of construct design, it is preferred to use the Release Factor-1 (RF1) deficient strain B95(DE3) ∆AfabRserB, though use of the standard RF1-containing BL21(DE3) ∆serB is also described. Both of these strains are serine auxotrophs and will not grow in standard minimal media. This protocol uses rich auto-induction media for streamlined and maximal production of homogeneously modified protein, yielding ~100–200 mg of single pSer-containing sfGFP per liter of culture. Using this genetic code expansion (GCE) approach, in which pSer is installed into proteins during translation, allows researchers to produce milligram quantities of specific phospho-proteins without requiring kinases, which can be purified for downstream in vitro studies relating to phosphorylation-dependent signaling systems, protein regulation by phosphorylation, and protein–protein interactions.


Graphical abstract:




Novel Protein Production Method Combining Native Expression in Human Cells with an Intein-based Affinity Purification and Self-cleavable Tag
Authors:  Peter J. Carman and Roberto Dominguez, date: 03/20/2022, view: 1541, Q&A: 0

The human proteins used in most biochemical studies are commonly obtained using bacterial expression. Owing to its relative simplicity and low cost, this approach has been extremely successful, but is inadequate for many proteins that require the mammalian folding machinery and posttranslational modifications (PTMs) for function. Moreover, the expressed proteins are typically purified using N- and/or C-terminal affinity tags, which are often left on proteins or leave non-native extra amino acids when removed proteolytically. Many proteins cannot tolerate such extra amino acids for function. Here we describe a protein production method that resolves both these issues. Our method combines expression in human Expi293F cells, which grow in suspension to high density and can process native PTMs, with a chitin-binding domain (CBD)-intein affinity purification and self-cleavable tag, which can be precisely removed after purification. In this protocol, we describe how to clone a target gene into our specifically designed human cell expression vector (pJCX4), and how to efficiently transfect the Expi293F cells and purify the expressed proteins using a chitin affinity resin.


Graphic abstract:



Characterising Plant Deubiquitinases with in vitro Activity-based Labelling and Ubiquitin Chain Disassembly Assays
Authors:  Michael J. Skelly and Steven H. Spoel, date: 05/05/2021, view: 3602, Q&A: 0

Post-translational modification of proteins by ubiquitin is an essential cellular signaling mechanism in all eukaryotes. Ubiquitin is removed from target proteins by a wide range of deubiquitinase (DUB) enzymes with different activities and substrate specificities. Understanding how DUBs function in vitro is a vital first step to uncovering their cellular roles. Here, we provide protocols for the rapid analysis of DUB activity in vitro by activity-based labelling with the suicide probe, HA-ubiquitin vinyl sulfone (HA-UbVS), and ubiquitin chain disassembly assays. We have previously used these methods to analyse the activity of the Arabidopsis thaliana DUB, UBP6, but in principle, these protocols are applicable to any DUB of interest.

Non-radioactive Assay to Determine Product Profile of Short-chain Isoprenyl Diphosphate Synthases
Authors:  Avanish Rai and Dinesh A. Nagegowda, date: 01/05/2021, view: 2600, Q&A: 0
Isoprenoids represent the largest class of metabolites with amazing diversities in structure and function. They are involved in protecting plants against pathogens or herbivores or involved in attracting pollinators. Isoprenoids are derived from geranyl diphosphate (GPP; C10), farnesyl diphosphate (FPP; C15), geranylgeranyl diphosphate (GGPP; C20), and geranylfarnesyl diphosphate (GFPP; C25) that are in turn formed by sequential condensations of isopentenyl diphosphate (IPP; C5) with an allylic acceptor such as dimethylallyl diphosphate (DMAPP; C5), GPP, FPP, or GGPP in a reaction catalyzed by isoprenyl diphosphate synthases (IDSs). IDS enzyme assay for determination of prenyl diphosphate products is generally performed using radiolabelled substrates, and the products formed are identified by employing expensive instruments such as phosphor imager, radio-GC, or radioHPLC. Though a non-radioactive assay for measuring IDS activity in crude plant extract has been reported, it requires a complex methodology utilizing chromatography coupled with tandem mass spectrometry (LC/MS-MS). Here, we describe a non-radioactive and simple inexpensive assay for determining the IDS assay products using non-radiolabeled IPP and its co-allylic substrates DMAPP,GPP, and FPP. The detection of prenyl diphosphate products generated in the assay was highly efficient and spots corresponding to prenyl alcohols were visible at >40 µM concentrations of IPP and DMAPP/GPP/FPP substrates. The protocol described here is sensitive, reliable, and technically simple, which could be used for functional characterization of IDS candidates.
Simple Method to Determine Protein Redox State in Arabidopsis thaliana
Authors:  Keisuke Yoshida and Toru Hisabori, date: 06/05/2019, view: 6117, Q&A: 1
Thiol-based redox regulation is a posttranslational protein modification that plays a key role in many biological aspects. To understand its regulatory functions, we need a method to directly assess protein redox state in vivo. Here we present a simple procedure to determine protein redox state in a model plant Arabidopsis thaliana. Our method consists of three key steps: (i) redox fixation by rapidly freezing plant tissues in the liquid nitrogen, (ii) labeling of thiol groups with the maleimide reagent, and (iii) protein detection by Western blotting. The redox state of a specific or given protein can be discriminated by the mobility change on SDS-PAGE with high sensitivity. This method provides a novel strategy to dissect the working dynamics of the redox-regulatory system in plants.
Ubiquitin Proteasome Activity Measurement in Total Plant Extracts
Authors:  Suayib Üstün and Frederik Börnke, date: 09/05/2017, view: 8350, Q&A: 0
The fine-tuned balance of protein level, conformation and location within the cell is vital for the dynamic changes required for a cell to respond to a given stimulus. This requires the regulated turnover of damaged or short-lived proteins through the ubiquitin proteasome system (UPS). Thus, the protease activity of the proteasome is adjusted to meet the current demands of protein degradation via the UPS within the cell. We describe the adaptation of an intramolecular quenched fluorescence assay utilizing substrate-mimic peptides for the measurement of proteasome activity in total plant extracts. The peptide substrates contain donor-quencher pairs that flank the scissile bond. Following cleavage, the increase in dequenched donor emission of the product is subsequently measured over time and used to calculate the relative proteasome activity.
PRODIGY: A Contact-based Predictor of Binding Affinity in Protein-protein Complexes
Authors:  Anna Vangone and Alexandre M. J. J. Bonvin, date: 02/05/2017, view: 12066, Q&A: 0
Biomolecular interactions between proteins regulate and control almost every biological process in the cell. Understanding these interactions is therefore a crucial step in the investigation of biological systems and in drug design. Many efforts have been devoted to unravel principles of protein-protein interactions. Recently, we introduced a simple but robust descriptor of binding affinity based only on structural properties of a protein-protein complex. In Vangone and Bonvin (2015), we demonstrated that the number of interfacial contacts at the interface of a protein-protein complex correlates with the experimental binding affinity. Our findings have led one of the best performing predictor so far reported (Pearson’s Correlation r = 0.73; RMSE = 1.89 kcal mol-1). Despite the importance of the topic, there is surprisingly only a limited number of online tools for fast and easy prediction of binding affinity. For this reason, we implemented our predictor into the user-friendly PRODIGY web-server. In this protocol, we explain the use of the PRODIGY web-server to predict the affinity of a protein-protein complex from its three-dimensional structure. The PRODIGY server is freely available at: http://milou.science.uu.nl/services/PRODIGY.
We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.