Hot Plasmids: Fall 2024

Every few months, we highlight some of the new plasmids, antibodies, and viral preps in the repository through our Hot Plasmids articles. 

Here's what you'll find in this post:

• Anti-V5 and other new epitope tag antibodies from IPI
• New tool for structural studies of E3 ligase cereblon
• Optimized mScarlet-based tags for live imaging in C. elegans
• CRISPR/Cas9 for gene disruption in M. abscessus 
• Bioluminescent reporter of mechanical forces

Epitope tag antibody collection grows

By Ashley Waldron

We are excited to highlight the Anti-V5 tag antibody produced by our partners at the Institute for Protein Innovation (IPI), part of a new collection of epitope tag antibodies available through Addgene.

Anti-V5 [IPI-SV5-Pk1] is a recombinant version of the original V5 tag mouse monoclonal antibody, which recognizes a short, linear epitope derived from the P and V proteins of Simian virus 5 (SV5) (Randall et al., 1987). The epitope (GKPIPNPLLGLDST) has been used as a tag since 1992 (Hanke et al., 1992), and has proven itself a versatile option for tagging proteins, as it can be fused to the N- or C-terminus (or even in the middle of proteins) and performs well in many different antibody-based applications. 

IPI’s recombinant version is a fusion of mouse variable and rabbit constant domains and is recommended for use in western blots (Figure 1). If you prefer to produce your own antibody or want to swap out the constant domains, the plasmids for the heavy chain and light chain are also available from Addgene!

Figure 1: Experimental validation for Anti-V5 [IPI-SV5-Pk1] in a western blot assay. Image reused from Anderson et al., 2024 under CC BY license. 

Find Anti-V5 [IPI-SV5-Pk1] Antibody here!

• Anderson, Z., Zhu, H., Riedel, T., Moshinsky, D., Meijers, R. (2024). Western Blot for Anti-V5 [IPI-SV5-Pk1]. Addgene Report, https://doi.org/10.57733/addgene.l0ulu4.
• Hanke, T., Szawlowski, P., & Randall, R. E. (1992). Construction of solid matrix-antibody-antigen complexes containing simian immunodeficiency virus p27 using tag-specific monoclonal antibody and tag-linked antigen. J Gen Virol., 73 (Pt 3), 653–660. https://doi.org/10.1099/0022-1317-73-3-653. PMID 1372038.
• Randall, R. E., Young, D. F., Goswami, K. K., & Russell, W. C. (1987). Isolation and characterization of monoclonal antibodies to simian virus 5 and their use in revealing antigenic differences between human, canine and simian isolates. J Gen Virol., 68 (Pt 11), 2769–2780. https://doi.org/10.1099/0022-1317-68-11-2769. PMID 2445904.

New tool for structural studies of E3 ligase cereblon

By Mike Lacy

Several types of drugs target the ubiquitin E3 ligase cereblon (CRBN), including popular new approaches like PROTAC targeted protein degraders. But CRBN has been difficult to study in vitro, requiring either expressing it with additional cofactors or using significantly truncated forms that limit its functionality. To make biophysical and structural studies more feasible, the lab of Alessio Ciulli and collaborators developed CRBN^midi (Kroupova et al., 2024). This construct contains the Lon and TBD domains, part of the HB domain, and several mutations to improve solubility and stability (Figure 2). 

Figure 2: Crystal structures of CRBN^midi alone (A) and in a ternary complex with the multiple myeloma drug mezigdomide and ubiquitination substrate Ikaros (IKZF1^ZF2) (B). Image adapted from Kroupova et al., 2024 under CC BY license.

This construct can be easily expressed and purified from E. coli, retains important functional properties, and is amenable to a wide range of biophysical assays (Kroupova et al., 2024). They were able to study and crystallize CRBN^midi on its own, with drug ligands, and in ternary complexes with other proteins, establishing CRBN^midi as a promising tool for future structure-guided drug design and studies of protein ubiquitination and degradation.

Find the CRBNmidi plasmid here!

• Kroupova, A. et al. (2024). Design of a Cereblon construct for crystallographic and biophysical studies of protein degraders. Nat Commun., 15(1), 8885. https://doi.org/10.1038/s41467-024-52871-9. PMID 39406745.

Optimizing mScarlet tags for live imaging in C. elegans

By Alyssa Neuhaus

Red fluorescent proteins like mScarlet are essential tools for visualizing biological processes in vivo. However, mScarlet’s slow maturation rate limits its use in systems like the C. elegans embryo, where developmental processes happen within a few hours. In order to use the latest mScarlet tags, mScarlet3 and mScarlet-I3, Oliver Hobert’s lab recently generated new constructs specifically for C. elegans (Cao et al., 2024).

The team designed coding sequences for mScarlet3 and mScarlet-I3 that were codon-optimized for C. elegans and included artificial introns to improve expression. When using these constructs to tag the GOLG-4 protein, mScarlet3 was 50% brighter than the original worm-optimized wrmScarlet. mScarlet-I3, while initially dimmer, showed greater photostability and also matured considerably faster, with expression levels comparable to GFP during early embryogenesis (Figure 3). mScarlet-I3 also offered clearer signals due to reduced background signal when tested in more highly autofluorescent tissues like the intestine.

Figure 3: Expression of fluorophore-tagged GOLG-4 through eight stages of embryonic development. Representative images of single focal planes through the middle of each embryo are shown, comparing GFP, wrmScarlet (mScarlet), and the new mScarlet3 and mScarlet-I3 constructs. Scale bar for all panels: 20 µm. Image reused from Cao et al., 2024 under CC-BY license. 

These optimized fluorescent proteins will be a useful addition to the toolkit for visualizing cellular processes in C. elegans: mScarlet3 for applications needing intense brightness (such as low-expression proteins of interest) and mScarlet-I3 for scenarios requiring rapid maturation and prolonged imaging with minimal background.

Find these C. elegans mScarlet plasmids here!

• Cao W.X., Merritt D.M., Pe K., Cesar M., Hobert O. (2024) Comparative analysis of new mScarlet-based red fluorescent tags in Caenorhabditis elegans. Genetics, 228(2), iyae126. https://doi.org/10.1093/genetics/iyae126.

Gene disruption in Mycobacterium abscessus using a dual-plasmid CRISPR/Cas9 system

By Aryana Khosravani

Deborah Hung’s lab has developed a novel dual-plasmid CRISPR/Cas9 system for gene disruption in Mycobacterium abscessus, an increasingly common human pathogen notorious for its high resistance to antibiotics. Several CRISPR/Cas-based systems have been developed for other mycobacteria, but such systems don’t always work across species due to differences in natural DNA repair pathways and spontaneous mutations. 

Here, the researchers were able to target multiple genes in M. abscessus either simultaneously or stepwise, achieving mutation efficiencies of up to about 9% of colonies (10² to 10⁴ times higher than traditional homologous recombination methods). By using mCherry for selection of sgRNA plasmid transformants, they avoid M. abscessus’ high rate of spontaneous antibiotic resistance interfering with selection. Using an inducible Cas9 expression and removing the sgRNA plasmid after identifying successful knockouts limits further cutting and errors (Figure 4).

This work marks the first use of CRISPR/Cas9 in M. abscessus. The simplicity and efficiency of this dual-plasmid approach makes it a valuable resource for advancing our understanding of its biology, pathogenicity, and resistance mechanisms.

Figure 4. Overview of the dual-plasmid CRISPR/Cas9 workflow in M. abscessus. A) Sth1Cas9 mediates double strand break at the target site, which results in either bacterial death or indels after DNA repair. B) M. abscessus is first transformed with Cas9 on an integrative plasmid containing tetracycline repressor TetR and resistance selection marker KanR. This strain is then transformed with the sgRNA cassette on a plasmid expressing mCherry and resistance selection marker HygR. The CRISPR system is induced to cause disruption of the target gene. Lastly, curing the mCherry-sgRNA plasmid leaves an edited strain carrying Cas9 to allow the introduction of additional sgRNAs. Image reused from Neo et al., 2024 under CC-BY license.

Find plasmids for CRISPR/Cas9 in M. abscessus here!

• Neo D, Clatworthy A, and Hung, D. (2024). A dual-plasmid CRISPR/Cas9-based method for rapid and efficient genetic disruption in Mycobacterium abscessus. J Bacteriol., 206(3), e0033523. https://doi.org/10.1128/jb.00335-23. 

Bioluminescent reporting of mechanical forces with PILATeS

By Emily P. Bentley

Time to get flexible! Alex Dunn’s lab and collaborators have deposited a new mechanosensor called PILATeS, Pulling-Induced Luciferase Association Tension Sensor (Zhong et al., 2024). In this new variation on their previous sensor STReTCh, the 11-amino acid BiT fragment of luciferase is embedded in the I10 domain of the human titin protein and only exposed when the module unfolds under tension. When this happens, BiT and the LgBiT luciferase fragment (expressed separately) assemble and catalyze the luminescence reaction (Figure 5). The researchers used the reporter to visualize biologically relevant forces at focal adhesions in living cells in several different contexts.

Figure 5: Left: Mechanism of the PILATeS tension sensor. The LgBiT (cyan) fragment of luciferase binds to the BiT Tag (also cyan) embedded in the Titin I10 protein (purple) only when Titin I10 unfolds under tension. Upon binding, LgBiT and BiT Tag catalyze a luminescent reaction with the substrate furimazine. Right: Demonstration of PILATeS. Cell adhesion to a PILATeS-functionalized coverslip creates mechanical tension, and the luminescence colocalizes with the focal adhesion marker paxillin (visualized with GFP). Reprinted with permission from Zhong et al, 2024. Copyright 2024 American Chemical Society.

Both PILATeS (18 kDa) and STReTCh (11 kDa) are significantly smaller than FRET-based mechanosensors (~60 kDa) and are compatible with common microscopy methods. Plus, the luminescence wavelength (460 nm) is shorter than many popular fluorescence markers like GFP (510 nm), allowing co-imaging. And because bioluminescent markers do not need to be excited by an external light source, they have lower background signal and can be used in light-sensitive systems. Most excitingly, assembly of the split luciferase is reversible, making PILATeS suitable for live imaging with a time resolution of minutes. 

Find PILATeS plasmids here!

• Zhong, B. L., Elliot, J. M., Wang, P., Li, H., Hall, R. N., Wang, B., Prakash, M., & Dunn, A. R. (2024). Split Luciferase Molecular Tension Sensors for Bioluminescent Readout of Mechanical Forces in Biological Systems. ACS Sensors, 9(7), 3489–3495. https://doi.org/10.1021/acssensors.3c02664.