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September 2014: New Channelrhodopsins, Tools for CRISPR gRNA Validation, & More
Shining a Light on Channelrhodopsins – Chronos & Chrimson
Through de novo-sequencing of 127 algal transcriptomes, as well as further optimization through engineering, Ed Boyden’s lab has identified a variety of new channelrhodopsins for use as optogenetics tools. These tools include a pair of channelrhodopsins, Chronos and Chrimson, which have enabled the group to perform independent two-color optical excitation of neurons.
The paper describes five previously unknown channelrhodopsins from different species: Stigleoclonium helveticum (ShChR), Chlamydomonas noctigama (CnChR1), Chloromonas subdivisa (CsChR), Chloromonas oogama (CoChR), and Scherffelia dubia (SdChR). ShChR, nicknamed Chronos, is blue and green light-excitable and has faster kinetics than those of other channelrhodopsins. CnChR1, nicknamed Chrimson, is the first reported yellow-peaked channelrhodopsin with a spectral peak at 590 nm, which is 45 nm more red-shifted compared to other variants. The group further optimized Chrimson through mutagenesis (K176R) to improve the otherwise slow off-kinetics (15.8 ± 0.4ms from 21.4 ± 1.1 ms); this new variant was named ChrimsonR. Plasmids containing Chronos, Chrimson, ChrimsonR, CsChR, and CoChR have been deposited to Addgene, including lentiviral and AAV expression vectors for certain variants.
Klapoetke et al., Nat Methods 2014 Mar 11;(3):338-46.
For more information and descriptions of various optogenetics plasmid tools, visit Addgene’s optogenetics guide.
More Optogenetics Tools – Light-activated Receptor Tyrosine Kinases
Receptor tyrosine kinases (RTKs) are a large class of cell-surface receptors which play a critical role in development and are often implicated in disease progression. One of the major challenges in signaling research is the inability to replicate the spatio-temporal precision with which signaling events occur in a physiological setting. Genetic techniques typically rely on overexpression or knock-down, where signaling dynamics are influenced by the relatively long life-cycle of a protein from expression to degradation. Chemical approaches may require expensive peptides or small molecules and exceed a physiologically relevant exposure in intensity and/or duration. Both of these strategies carry the risk of off-target effects.
Harald Janovjak and his team at the Institute of Science and Technology Austria, decided to take a novel approach to control for these effects, drawing on the rapidly growing field of optogenetics. Using a rational protein engineering approach, they designed Opto-RTKs, which activate signaling cascades on exposure to low-intensity blue light. This approach relies on the incorporation of light-oxygen-voltage (LOV) sensing domains from algae into chimeric Opto-RTKs. The LOV domains bind flavin cofactors and dimerize on exposure to light, bringing the intracellular kinase domains into contact and initiating signaling. Grusch et al. demonstrate that Opto-RTKs have a similar level of background activity and activation as their wild-type counterparts.
The Janovjak lab has deposited their 3 Opto-RTK constructs – Opto-mFGFR1, Opto-hEGFR, and Opto-hRET – as well as various LOV-domain-mVenus constructs used in the study for those who would like to extend this work into their RTK of interest.
Grusch, M. et al., EMBO J 2014 Aug 1;33(15):1713-26.
RUSH & CRUSH – Rapid & Conditional Gene Silencing in RNAi Transgenic Mice
The Jackson-Grusby lab has designed two vectors for use in conditional and reversible gene silencing in RNAi transgenic mouse models and embryonic stem (ES) cells. These vectors, termed RUSH (For ROSA26 U6 short hairpin) and CRUSH (Conditional RUSH) use Cre-mediated recombination to turn on or off the expression of shRNA. This system requires less work than using established methods and minimizes some of the technical issues associated with high levels of shRNA expression. The capability to turn RUSH and CRUSH alleles off or on enables this method to rapidly address questions of tissue-specificity and cell autonomy of gene function in development. To validate the use of the CRUSH vector in transgenic mice, the lab established a dual-color RNAi “sensor” mouse strain in which Cre expression causes the induction of both DsRed fluorescence and GFP shRNA. Mice carrying the R26DsRedR; CRUSH-GFP; and Nestin-Cre alleles showed efficient GFP knockdown in eyes and clonogenic neural stem cells concomitant with activated DsRed2 expression.
Brown et al., Genesis 2014 Jan;52(1):39-48.
A New Tool for CRISPR gRNA Validation
Selecting a gRNA sequence that effectively targets your gene/region of interest is a key step for CRISPR/Cas9 gene editing. Dr. Masahito Ikawa has created a GFP reporter plasmid for scientists to validate the efficacy of their gRNAs.
The first step is to clone the target sequence in between two fragments of EGFP in pCAG-EGxxFP. The EGFP fragments contain 482bp of overlapping sequence that direct Homologous Recombination (HR) or Single Strand Annealing (SSA) in the event of a DNA break. Next, the gRNA being tested is expressed along with Cas9. If the gRNA effectively cuts the target sequence, the plasmid undergoes HR or SSA to reconstitute functional GFP, and the cells will turn green.
Mashiko et al., Sci Rep 2013 Nov 27; 3:3355.
For more CRISPR plasmids and resources, visit Addgene's frequently updated CRISPR/Cas9 guide.
Irreversible Peptide-Peptide Ligation Using SpyLigase
Building off their SpyTag/SpyCatcher system for protein-peptide locking, Mark Howarth’s lab has developed a new tool for peptide-peptide locking. The new technology is known as SpyLigase and is a protein domain that promotes the formation of an isopeptide bond between 2 peptide tags, SpyTag and KTag. The group demonstrated the use of the SpyLigase peptide-peptide interaction to link affibodies or antibodies against common tumor markers to enhance cancer cell capture.
For further reading about SpyLigase technology, read the Addgene interview with Mark Howarth.
Rinehart lab reagents for improved expression of recombinant phosphoproteins
Protein phosphorylation is one of the most abundant forms of posttranslational modifications in cells and research into its many roles in protein function and signaling networks continues to expand. The labs of Jesse Rinehart and Dieter Söll at Yale University previously changed the way researchers can explore important questions surrounding serine phosphorylation by adding this phosphorylated amino acid to the genetic code of E. coli (Park et al., Science 2011).
The Rinehart lab has now made improvements to this system by engineering cells that lack release factor one (RF-1; Bacterial strain EcAR7) and minimizing the set of plasmids required to make singly or multiply phosphorylated proteins (B40 OTS and pCRT7-GFP). To demonstrate the improvements of the system, the Rinehart lab synthesized the activated form of human mitogen-activated ERK activating kinase 1 (MEK1) with either one or two phosphoserine residues cotranslationally inserted in their canonical positions (SP218, SP222) using the original or improved phosphoprotein synthesis reagents. One phosphoserine (SP218) insertion was moderately enhanced while two phosphoserine insertions (SP218/SP222) was dramatically enhanced with the improved system. This MEK vector is also available at Addgene and can be used as a control for your experiments. For more information, please see Addgene’s information page for the Rinehart phosphoprotein system, which includes detailed protocols provided by the Rinehart lab.
pOSIP and the Clonetegration
Classic genetic engineering methods enabling chromosomal integration of sequences in bacteria are time-consuming and involve many steps. The Drew Endy and Keith Shearwin labs have developed a new, streamlined approach to genetic engineering which drastically reduces the time and effort needed to insert new genes into bacteria. They designed the pOSIP (one-step integration plasmid) series of plasmids, vectors that convey both the sequence to be integrated and a removable integrase cassette. They validated this methodology in two differents types of bacteria (E. coli and Salmonella typhimurium) by integrating DNA sequences either sequentially or simultaneously.
This method, called by the authors the “Clonetegration”, is quick and easy to do. Clonetegration could become a “valuable technique facilitating genetic engineering with difficult-to-clone sequences and rapid construction of synthetic biological systems” as they predict. The pOSIP plasmid kit can be found at Addgene, so what are you waiting for? Start building up your own designer bacteria.
St-Pierre et al., ACS Synth Biol 2013 Sep 20;2(9):537-41.
June 2014: MoClo & Platinum Gate Kits, Davidson Lab FPs, CRISPRs, & More
DREADD-based Chemogenetic Technologies
After several years of distributing his Designer Receptors Exclusively Activated by Designer Drugs (DREADD) plasmids on his own, UNC-Chapel Hill's Bryan Roth has now deposited many of these constructs with Addgene. These G-protein coupled receptors have been engineered by the Roth lab to be activated by otherwise pharmacologically inert drug-like small molecules, allowing labs to precisely and non-invasively control neuronal signaling.
For more information on DREADDs, please see the Roth lab's DREADD users blog.
Michael Davidson Lab Fluorescent Protein Collection
Michael Davidson and his lab from Florida State University have contributed their comprehensive collection of ORFs tagged with a variety of fluorescent proteins. In addition to this collection of ORFs, over 100 empty backbones are available from the Davidson lab for tagging your gene of interest.
The Davidson plasmid collection includes excitation/emission, localization, sequence, and supplemental information for many of the plasmids so you can easily find what you need.
We currently have ~ 300 plasmids (empty backbones and mEmerald tagged ORFs) available for request, with more plasmids becoming available everyday. Browse the collection here.
For more information on these tools, visit Davidson's Molecular Expressions website for additional images, tutorials, optical microscopy protocols, and many more resources.
Looking for other fluorescent proteins? Find more at Addgene’s Fluorescent Protein Guide.
New CRISPR Plasmids Available!
New Lentiviral CRISPR activator and repressor plasmids from Scot Wolfe's lab. These include Tet-inducible CRISPR activators and repressor plasmids. (Kearns et al., Development. 2014..)
CRISPRs for Xenopus! From the lab of Yonglong Chen, pCS2-3xFLAG-NLS-SpCas9-NLS is a Cas9 expression plasmid that was used by Chen and colleagues for genome editing in Xenopus tropicalis. (Guo et al., Development. 2014..)
A new, higher specificity genome editing system that combines TALENs and CRISPRS. Developed by David Liu and colleagues, FokI-dCas9 expresses Fok1 nuclease domain fused to catalytically inactive Cas9 DNA-binding domain in mammalian cells. (Guilinger et al., Nat Biotechnol. 2014..)
Our CRISPR-Cas collection of plasmids updates frequently, so visit our CRISPR pages often to find the most recently deposited tools, resources, protocols, and more.
MoClo Modular Cloning System
Click on image to see full figure and more information about the MoClo Kits available at Addgene. Image from Weber et al., PLoS One. 2011 Feb 18;6(2):e16765.
Synthetic biologists have developed a modular cloning strategy, MoClo, which uses the Type IIS restriction enzymes BsaI and BpiI/BbsI to efficiently assemble up to six DNA fragments at a time. This method (based on the Golden Gate technology) exploits the ability of Type IIS enzymes to cut outside their recognition site, and permits DNA fragments with compatible overhangs to be efficiently assembled. Scientists can engineer unique enzyme recognition sites that flank a DNA module in an inverse orientation, so that multiple DNA components can directionally assemble in a single reaction, while retaining only a defined 4bp fusion site in between.
The MoClo system is comprised of three sets of cloning vectors (Level 0, 1, or 2) which can be utilized in three successive assembly steps. Before beginning, scientists can insert fragments of DNA containing basic parts (promoters, UTRs, coding sequences, terminators, etc) into individual Level 0 plasmids, or choose from a growing number of libraries containing pre-constructed standardized modules. In the first assembly step, compatible Level 0 vectors are directionally assembled into a Level 1 vector creating a single transcriptional unit (Ex: a promoter, 5’UTR, coding region, and terminator). Next, up to six Level 1 modules can be similarly assembled into a Level 2 vector, thus forming a functional genetic circuit. Flexibility has been built into the Level 2 vectors to allow for additional iterations of Level 1 assembly if necessary. Combining multiple Level 2 vectors in the final assembly step permits the creation of more complex constructs constrained only by the ability of E. coli to maintain the final plasmid after transformation.
Addgene depositors Sylvestre Marillonnet and Nicola Patron have assembled two collections of standardized genetic modules compatible with the MoClo system. The MoClo Toolkit provided by the Marillonnet Lab can be used to assembly general eukaryotic multigene constructs, while the Patron Lab MoClo Plant Parts kit contains modules specific for plant transformation.
Weber et al., PLoS One. 2011. Feb 18;6(2):e16765.
Engler et al., ACS Synth Biol 2014. Feb 5 (Web); DOI: 10.1021/sb4001504.
For further reading about Nicola Patron's MoClo kit and her plant synbio research, read the Addgene interview.
Engineering TALENs containing variable-repeats using Platinum Gate system
Interested in optimizing TALEN assembly and activity? The laboratory of Takashi Yamamoto has created a complete TALEN assembly system after systematically analyzing the effect of both the TALE scaffold and module on TALEN activity in a single-strand annealing (SSA) assay.
This new Platinum Gate TALEN Kit utilizes a 4-module assembly system, which reduces the number of individual repeat-variable di-residue (RVD) module plasmids and simultaneously increases the success rate of module assembly in the first Golden Gate reaction. While the DNA-binding specificity is imparted by the RVD at residues 12 and 13 in the TALE repeat, other naturally-occurring variations in the TALE repeat, referred to as “non-RVD variations”, were found to improve TALEN activity. Each positional group of module plasmids (ex. p1HD, p1NG, p1NI & p1NN) in the Platinum Gate system contains an identical variable repeat (VR) at the 4th and 32nd residues of the TALE repeat and the VR differs between the positional groups. Eight final destination vectors, consisting of all four final RVD modules in each of two different TALE scaffolds are provided, as the optimal scaffold can depend on the length of the spacer region between the target sequences. These updates to TALEN genome engineering demonstrate improved efficiency over previous reports.
Click on image to see full figure and more information about thePlatinum Gate TALEN Kit. Figure from Sakuma et al., Sci Rep (2013).
For additional information on using the Platinum Gate TALEN Kit, please see the Yamamoto lab’s protocol for TALEN construction.
Sakuma et al., Sci Rep. 2013. Nov 29;3:3379.
Interested in more genome engineering technologies? Browse others on Addgene's Genome Engineering Guide.
March 2014: New Neuronal Imaging Tools, GreenGate Cloning System, & More
Fire Up Those Neurons: mGRASP
A Nature 2012 article by Jinny Kim and colleagues describes their efforts to map the location and distribution of synapses in the mouse brain. Kim et al is utilizing a mammalian GRASP (GFP reconstitution across synaptic partners) technique based on functional complementation between two non-fluorescent split GFP fragments. When the two fragments, expressed in the presynaptic region of one neuron and postsynaptic region of a different neuron, come into proximity in the synaptic cleft, functional fluorescent GFP is reconstituted in vivo. Currently available from the Kim lab are 2 presynaptic and 2 postsynaptic targeting mGRASP plasmids. Additionally, the lab recently described the use of another set of mGRASP plasmids in their Neuron 2014 paper.
Next-Gen Brainbow Toolkit for Neuronal Imaging
Joshua Sanes and his team at the Center for Brain Science at Harvard University have developed a next-generation Brainbow toolkit for high-resolution fluorescent imaging of individual neurons. The technology generates a unique spectral identity for each cell in a population by expressing a randomly generated mix of fluorescent proteins, determined by competing recombination events at the genetic level. Upon Cre/loxP recombination, each transgene expresses one of three possible fluorescent proteins, chosen for minimal spectral overlap, minimal protein aggregation, and high photostabilty. When multiple cassettes are integrated, each recombines independently, generating tens or hundreds of possible combinations (depending on the number of copies). This facilitates the distinction of neighboring cells in imaging applications and the mapping of neuronal projections to their associated cell bodies.
The first versions of the Brainbow system were reported in 2007. In order to extend the utility of the system, the researchers developed three new versions. Flipbow employs the Flp recombinase/FRT system in place of Brainbow’s Cre/loxP, allowing for simultaneous use of the two systems in different tissues of the same animal. Flipbow additionally incorporates SUMO tags in the FP sequence for separation from Cre-based Brainbow-expressing cells. Autobow plasmids are all-in-one versions which express self-excising Cre recombinase from the same transgene as the Brainbow cassette, simplifying experiments when additional cross-breeding steps are undesired or infeasible. Finally, an adeno-associated viral (AAV) system enables greater spatio-temporal control over expression and increases the number of species in which Brainbow may be used. This system uses two plasmids in tandem, with Cre-dependent inversion determining between 0 and 2 FPs expressed from each copy. Brainbow, Flipbow, and Autobow systems are available with either the Thy1 or CAG promoter, while Brainbow AAV is under control of the EF1a promoter.
pCoofy Vectors for Optimizing Protein Expression
Under the direction of Sabine Suppmann, the Recombinant Protein Production group at Max-Planck Institute of Biochemistry has developed a number of expression vectors for use with Sequence and Ligation Independent Cloning (SLIC). The pCoofy series of plasmids contain a variety of N- and C-terminal tags (including His, S-tag, OneStrep, CBP, Trx, GST, Halo, MBP, NusA and SUMO) for optimizing expression, solubilization and purification and have been tested in bacterial, insect and mammalian cells. These vectors were designed for parallel testing and screening of constructs in multiple host cells in order to optimize expression. The expression plasmids for a given species are based on the same backbone to permit expression levels to be directly compared amongst the different tags
To clone a sequence or gene of interest into the pCoofy vectors, select the appropriate pair of vector and gene primers from Table 2 of the associated publication which contain regions of sequence homology between the vector and gene primer for SLIC cloning. Amplify the pCoofy vector and the sequence of gene of interest in separate PCR amplication reactions for recombination in SLIC cloning to form the desired vector. The pCoofy vectors contain the ccdB cassette, which is not copied during the PCR amplification step, so that only vectors with the desired sequence of interest are retained after SLIC cloning. The general cloning strategy described in the associated publication can be used to generate any combination of tags for optimizing protein expression and purification in a fast, efficient and affordable way.
Scholz et al.., BMC Biotechnology. 2013. Feb 14;13:12.
Image from Scholz et al.., BMC Biotechnology. 2013. Feb 14;13:12.
GreenGate Cloning System for Plant Transgenesis
Developed by Jan Lohmann and colleagues, GreenGate is a cloning system for the rapid assembly of plant transformation constructs. As the name suggests, GreenGate is based on the Golden Gate cloning method, but has been modified specifically to improve plant transgenesis. The GreenGate kit available at Addgene includes six individual types of pre-cloned insert modules (plant promoter, N-terminal tag, coding sequence of the gene of interest, C-terminal tag, plant terminator, and plant resistance cassette) in pUC19 based entry vectors, as well as the pGreen-IIS based destination vectors.
To learn more about the GreenGate cloning system, see the detailed plasmid kit page or read our blog post: Quick, Versatile Plant Transgenesis with GreenGate Plasmids.
Lampropoulos et al., PLoS One. 2013. Dec 20;8(12):e83043.
Lentiviral CRISPR Libraries for Knockout Screening
New systems have been developed and deposited with Addgene which allow scientists to use CRISPR-Cas technology to perform genome-wide knockout screens. These vectors and sgRNA libraries expand upon the CRISPR family of plasmids by offering a lentivirus-based mechanism for sgRNA delivery and providing a means for large scale functional screens.
For more information on these new CRISPR screening tools, see our CRISPR/Cas Plasmids: Pooled Libraries webpage or read our blog post, Lentiviral CRISPR Libraries Enable Genome-Scale, Knockout Screening.
Also, check out our updated CRISPR-Cas resources at www.addgene.org/CRISPR/ to browse CRISPR plasmids, watch informational videos, download protocols, and more! Looking for backgroubnd information on CRISPR technology? See our improved CRISPR-Cas Guide.
December 2013: Light Controlled Genome Editing, Hydrogen Peroxide Sensor, CRISPRs, & More
Affinity and Fluorescent Protein Tagged Bacterial Expression Vectors
Looking for bacterial expression vectors with affinity tags for purification or fluorescent reporter gene fusions? The laboratory of Thorben Dammeyer constructed a set of plasmids with several combinations of affinity tags and fluorescent YFP-fusion proteins for periplasmic and cytoplasmic expression.
The plasmids in the pTD series share a broad host range RK2 origin of replication and a strong, IPTG-inducible lacIq-Ptrc promoter. Variations in the presence and location of Strep, Twin-Strep, and His tags allow for affinity purifications or co-purifications, while an optional pelB signal sequence utilizes the secretory pathway for export to the periplasmic space. A synthetically engineered EYFP variant was created to permit expression of a mature, active EYFP in the periplasm of Gram negative bacteria such as E. coli and P. putida, as standard EGFP and EYFP are unable to mature in the periplasm after export by the secretory pathway. These plasmids are based on the Standard European Vector Architecture (SEVA) platform to permit exchange of the origin of replication, promoter/MCS and antibiotic resistance modules with other SEVA compatible modules.
Dammeyer et al., Microb Cell Fact. 2013. May 20;12(1):49.
Light Controlled Genome Editing: LITE
Optogenetics meets genome editing in the newest tools developed by the lab of Feng Zhang. These light-inducible transcriptional effectors (LITEs) are designed to bind specific genes and turn them on or off in response to light. These LITEs have been packaged in viral vectors and can be targeted to specific cell populations. Konermann et al. demonstrated the use of these tools to control gene expression in mouse neurons and in the brains of living mice.
For more about these new optogenetic tools, check out our blog post: Let There Be LITE Plasmids.
Konermann et al., Nature. 2013. Aug 22; 500: 472–476.
HyPer3: Fluorescent Protein Sensor for Reactive Oxygen Species
The lab of Vsevolod Belousov has deposited their latest hydrogen peroxide sensor, HyPer3. In the presence of reactive oxygen species (ROS), the excitation properties of HyPer3 change such that the intensity of the emitted light (516 nm) from 500 nm excitation increases relative to that emitted by 420 nm excitation (increase in 500/420 ratio). The new generation sensor has greater dynamic range than HyPer1, faster response times than HyPer2, and is available for either mammalian or bacterial expression. Use it to track ROS changes in real time by fluorescence imaging.
Bilan et al., ACS Chem Biol. 2013. Mar 15; 8(3):535-42.
Latest CRISPR-Cas9 Plasmids
The genome engineering technology known as CRISPR/Cas has recently been utilized in exciting new ways.
Scientists have devised ways to harness Cas9 nuclease to activate or repress genes. This was accomplished by fusing known transcriptional activator proteins (example VP64) or repressor proteins (example KRAB domain) to a catalytically inactive Cas9 nuclease. When targeted to promoter regions by a specific gRNA, these activator or repressor proteins have been shown to up or down regulate gene expression.
Additionally, multiple research groups have recently identified and utilized the type II CRISPR/Cas systems from several different bacterial species. For reference, the original discovery and application of CRISPR genome engineering technology utilized the type II CRISPR/Cas system from Streptococcus pyogenes. The key difference between these new CRISPR systems is the unique PAM (Protospacer-Adjacent Motif) sequence recognized by the Cas9 nuclease in each species. Different PAM sequences allow for an increase in potential target sites for gRNAs (a gRNA can be targeted to any sequence in the genome that ends with an appropriate PAM sequence). Different PAM sequences also allow for multiple, simultaneous CRISPR/Cas driven genome manipulations. For example, a cutting Cas9 from S. pyogenes and an activating Cas9 from N. meningitidis can function within the same cell, at the same time, without interfering with one another. These advances add increased functionality to the already versatile system that is CRISPR.
Interested in reading about the history of CRISPR-Cas technology? Checkout our blog post: History of CRISPR Cas - A tale of survival and evolution.
pDusk and pDawn: Light Regulated Bacterial Expression Plasmids
Building on advances in optogenetics, Andreas Mӧglich's lab has built pDusk and pDawn, two complementary plasmids for light regulated expression of recombinant proteins in E. Coli. These plasmids rely on the engineered two-component regulatory system YF1/FixJ. YF1 is a synthetic, photosensitive kinase, which uses the ubiquitous flavin mononucleotide as its chromophore, and phosphorylates the transcriptional activator FixJ in the absence of blue light. Phosphorylated FixJ is able to drive high levels of gene expression from the FixK2 promoter. These elements are the basis for pDusk, which allows for insertion of your gene of interest directly downstream of the FixK2 promoter. Genes cloned into the multiple-cloning site (MCS) of pDusk will be expressed in the absence of blue or ambient (white) light, and expression levels can be varied with light intensity.
Ohlendorf et al., J Mol Biol. 2012. Mar 2, 416(4):534-42.
The complementary plasmid pDawn is a light-activated expression system, and may be used in combination with pDusk in experiments where alternating expression of different genes is desired. pDawn contains all of the elements of pDusk, except that phosphorylated FixJ now drives expression of the λ phage repressor cI, which in turn represses gene expression from the λ promoter pR, located upstream of the MCS. Because of the strength of both the λ phage repressor and promoter, pDawn has both lower background expression and better induction than pDusk, making it the better choice for preparative expression. After transformation, cultures can be grown to the desired density in the dark, and induced for expression by exposure to blue or ambient light. This obviates the need for chemical inducers such as IPTG, saving money and reducing potential exposures to contaminants. A big advantage of light vs. chemical induction is the ability to turn off expression by removing the light source.
Ohlendorf et al., J Mol Biol. 2012. Mar 2, 416(4):534-42.
Looking for other plasmids for optogenetics research? Check out Addgene’s Optogenetics Guide.
September 2013: Tools for Proteomic Mapping, NIR Fluorescent Probes, Newest TALEN Kit, & More
Tool for Proteomic Mapping of Mitochondria in Living Cells
Alice Ting's lab has designed a new technology for creating a spatially and temporally resolved proteomic map of large numbers of proteins in living cells. The method works by targeting ascorbate peroxidase (APEX) within the cell, resulting in biotinylation of nearby proteins. The biotinylated proteins are then analyzed by mass spectrometry, providing a readout of colocalized proteins from live cells.
The Ting Lab validated this technique by localizing APEX to the mitochondrial matrix with the plasmid pcDNA3-mito-APEX. This resulted in the identification of 495 proteins within the human mitochondrial matrix, including 31 which were not previously linked to the mitochondria. Browse the relevant plasmids.
Rhee et al., Science. 2013. Mar 15; 339(6125):1328-31.
Gateway-compatible Cloning and Expression Vectors
Have you ever wished that your favorite empty vector or tag was available in a Gateway-compatible version? We may have exactly what you are looking for already in our repository.
Image from Dubin et al., Plant Methods (Biomed Central). 2008. Jan 22; 4:3.
The lab of Giovanna Benvenuto created a series of twelve Gateway Entry vectors with six commonly used tags for either N- or C-terminal fusions. The available tags (STREP, HA, MYC, GST, ECFP and EYFP) are present within the attachment sites, avoiding any extra linker amino acids between the tag and insert. Traditional restriction enzyme cloning is used to insert a gene of interest into these Entry vectors, followed by recombination with a Gateway-compatible Destination vector of your choice for bacterial, insect, mammalian, plant or yeast expression. All of the vectors utilize the same cloning sites and contain a stop codon before the final attachment site in the entry cassette.
Converting existing vectors to Gateway-compatible vectors that can be used with recombination-based cloning can be a tedious process. Fortunately, the Yu-Zhu Zhang laboratory has developed a method using site-specific recombination to convert non-Gateway based vectors to Gateway-compatible vectors. This process was then used to create eleven Gateway-compatible Destination vectors from commonly used conventional empty vectors containing His, hemoglobin, EGFP, Flag, Myc-His tags or from other empty vectors used in adenoviral, bacterial, mammalian or yeast systems. An Entry vector containing your gene of interest can be recombined with one of these Gateway-compatible Destination vectors to generate an expression-ready construct.
More empty backbones can be found at Addgene’s Empty Backbones Guide.
New NIR Fluorescent Probes: iRFPs, PAiRFPs, and iSplit
Several types of new near-infrared fluorescent proteins derived from bacterial phytochrome photoreceptors (BphPs) have been developed by Vladislav Verkhusha’s lab and can be used for deep-tissue optical in vivo imaging. They fluoresce in mammalian cells and tissues without adding exogenous biliverdin. The four new spectrally distinct permanently fluorescent iRFP variants (iRFP670, iRFP682, iRFP702, and iRFP720) described by Shcherbakova et al., along with the group’s original iRFP (iRFP713), were shown to have high effective brightness and allowed multicolor imaging. Next, Piatkevich et al. described the engineering of photo-activatable iRFPs (PAiRFP1 and PAiRFP2), which can be ‘turned on’ by non-phototoxic far-red light and used for spatially selective imaging of tissues in living animals. Most recently, further development of the original iRFP resulted in a split fluorescence complementation probe, iSplit, by Filonov et al. iSplit was tested both in vitro and in vivo as a biomolecular fluorescence complementation (BiFC) reporter to detect protein-protein interactions.
Shcherbakova et al., Nat Methods. 2013. Jun 16; 10(8):751-4.
Piatkevich et al., Nat Commun. 2013. Jul 10; 4:2153.
Filonov et al., Chem Biol. 2013. Aug 22; 20(8):1078-86.
Looking for other fluorescent proteins? Check out Addgene’s Fluorescent Protein Guide.
The Open Source Wnt Project
Wnt signalling pathways play essential roles in embryonic development as well as tissue homoeostasis in adults, and their aberrant regulation has been linked to diseases in man including diabetes, neurodegeneration and cancer. In order to allow direct side by side comparison of the various mammalian Wnts and their function, the labs of Marian Waterman and David Virshup have developed a standardized set of Wnt expression plasmids. The kit contains the ORFs of all 19 human Wnts in the same expression backbone. Each ORF is cloned into 2 entry backbones, pENTR/D-TOPO with and without a STOP codon, and 2 mammalian expression backbones, with and without a C-terminal V5 tag. In addition, the Xi He lab has contributed a fifth set containing a modified version of each tagged ORF that enables epitope tagging without loss of Wnt signaling activity.
Najdi et al., Differentiation. 2012. Sep; 84(2):203-13.
Learn more about how the Open Source Wnt Kit was developed using crowd-sourcing in our interview with Dr. Marian Waterman.
Genome Engineering in hPSCs: New Musunuru/Cowan TALEN Kit
Developed by the labs of Kiran Musunuru and Chad Cowan, the newest TALEN kit allows for the quick and easy delivery of TALENs into human pluripotent stem cells and other difficult-to-transfect mammalian cell types. TALEN construction can be completed in 1-2 days without PCR amplification.
- Gene knockout by indel mutation induction
- Reporter line generation
- Correcting causal mutation in iPSC lines
- And more!
Ding et al., Cell Stem Cell. 2013. Feb 7;12(2):238-51.
Check out the Latest CRISPR Plasmids
Do you want to edit plant, fly, worm, or fish genomes? We now have CRISPRs for that! Are you interested in activating your gene of interest? New CRISPR technology lets you selectively activate your gene of choice!
Kamoun Lab: Using CRISPRs to modify plant genomes.
“Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease.” Nat Biotechnol. 2013.
Joung Lab: A new use of the CRISPR/Cas9 system to target and activate specific genes.
“CRISPR RNA-guided activation of endogenous human genes.” Nat Methods. 2013.
Chen and Wente Labs: Using CRISPRs to modify the zebrafish genome.
“Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system.” PNAS. 2013.
Calarco Lab: Using CRISPRs to modify the C. elegans genome.
“Heritable genome editing in C. elegans via a CRISPR-Cas9 system.” Nat Methods. 2013.
O’Connor-Giles, Wildonger, and Harrison Labs: Using CRISPRs to modify Drosophila genome.
“Genome Engineering of Drosophila with the CRISPR RNA-Guided Cas9 Nuclease.” Genetics. 2013.
Goldstein Lab: Using CRISPRs to modify the C. elegans genome.
“Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination.” Nat Methods. 2013.
Don't see the CRISPR/Cas system you're looking for here? Find more CRISPR/Cas9 plasmids at Addgene.
June 2013: Fluorescent Protein Kit, CRISPRi, New Vectors for Use with Golden Gate TALEN Kit
New Plasmid Kit: Fluorescent Proteins
Organizing your fluorophore combinations at the start of a research project can be a tricky task. Many times, having access to additional colors or combinations can be a big asset. The Hamdoun lab constructed a number of fluorescent plasmids for their recent JBC publication, and the fluorescent protein- containing empty vectors have wide applications for use in Zebrafish, Sea urchin, Xenopus, and C. elegans. The following 9 plasmids have been bundled together to provide a useful “starter kit” for screening fluorescent protein fusion expression in organisms or cells in which an exogenous mRNA can be injected and expressed. The utility of this kit is that it enables the user to generate N and C terminal fusions to mCherry, Cerulean, mCitrine, or EGFP, and could also be employed for many types of multicolor experiments or assays with fluorescent small organic molecules.
Gokirmak et al., J Biol Chem. 2012. Dec 21; 287(52):43876-83E.
CRISPRi: Repurposing of the CRISPR/Cas Genome-Editing Technology for Transcriptional Silencing
The CRISPR/Cas system is quickly becoming well known as an effective genome-editing technology, and a simple alternative to TALENs. With only two components, CRISPR systems utilize Cas9 nuclease to cleave DNA and chimeric guide RNA (gRNA) to target the Cas9 to a specific region of the genome. The system has been optimized for DNA editing in a variety of different species and cell types. The current technology utilizes Cas9’s nuclease abilities to destroy a specific locus in DNA through directed cleavage followed by non-homologous end joining (NHEJ). Alternatively, a modified Cas9 can be made to nick the DNA, cutting only one of the DNA strands to facilitate DNA replacement by homologous recombination. This requires the transfection of an additional plasmid that contains a complimentary sequence.
The Stanley Qi lab has created a new function of the CRISPR/Cas system called CRISPR Interference. CRISPRi is the latest tool available to scientists looking to manipulate the genome of their favorite organism. CRISPRi utilizes a catalytically inactive Cas9 nuclease in complex with a gRNA to interfere with transcription of the DNA downstream of its binding site. The mechanism of the interference is believed to involve physically impairing the RNA polymerase progression past the Cas9:gRNA complex. These CRISPRi plasmids are optimized for use in human cells and an additional set of CRISPRi plasmids are optimized for use in bacteria.
Qi et al., Cell. 2013. Feb 28;152(5):1173-83.
Alternative TALEN Assembly and Validation Vectors for Golden Gate TALEN Kit
Recent advances in genome editing technology, such as transcription activator-like effector nuclease (TALEN) systems, have reduced the barrier to studying gene function. With a focus on efficiency, the laboratory of Takashi Yamamoto has developed optimized array and destination vectors for use in combination with original vectors from the Golden Gate TALEN and TAL Effector kit, deposited by the labs of Dan Voytas and Adam Bogdanove. The modified pFUS array vectors are designed for six-module assembly to improve the assembly success rate and efficiency of the first Golden Gate cloning step, while reducing the number of necessary RVD module vectors. An optional ligation with pre-digested RVD and array vectors for the first module assembly step is reportedly more robust than the typical Golden Gate assembly method. The mammalian destination vectors offer the choice of a CMV/T7 or CAG promoter with codon-optimized FokI and Flag tag, ready for use in mammalian cells without additional cloning.
A novel TALEN evaluation system utilizing the pGL4-SSA vector included in the Yamamoto Lab TALEN Accessory Pack allows for validation of TALEN plasmids in mammalian cells using a luciferase reporter containing custom oligonucleotides corresponding to the TALEN target sequence in a single-strand annealing (SSA) assay. The Yamamoto lab has provided a step-by-step protocol describing the use of their array and destination vectors, as well as this universal TALEN validation assay in mammalian cells.
Sakuma et al., Genes Cells. 2013. Apr;18(4):315-26.
FREQ-Seq: a rapid method to determine specific allele frequencies from mixed populations.
To help scientists track the frequencies of specific alleles in microbial populations through time Christopher Marx's lab has engineered and validated a new method called FREQ-Seq. This strategy allows scientists to construct barcoded, locus-spe cific libraries compatible with Illumina next generation sequencing in order to study evolutionary dynamics. By counting DNA sequence reads, FREQ-Seq can quantitatively determine allele frequencies across timepoints or populations. This kit consists of a 48 plasmid adaptor library, with each plasmid carrying a unique barcoded Illumina-M13F bridging primer. These bridging primers are amplified and used to generate Illumina sequencing libraries using a 2-step PCR-based protocol and are compatible with single-end or paired-end read flow cells. FREQ-Seq is an open source platform and the libraries can be generated in a cost-effective manner with minimal bias.
Chubiz et al., PLoS One. 2012.; 7(10): e47959. doi:10.1371/journal.pone.0047959.
Linearly Tunable Gene Expression Systems
Experimental dose–response data for htetR::NLS::eGFP and mCherry expression in the two gene mammalian linearizer system plotted on log-log scale after background subtraction (defined as the fluorescence at 0 ng/mL of doxycycline).
Gábor Balázsi’s lab has developed a series of systems that allow for tunable gene expression. The group first developed the system in yeast by creating a synthetic linearizer gene circuit that controlled gene expression with a linear dependency on the extracellular concentration of an inducer (anhydrotetracycline). The use of a negative feedback circuit also resulted in gene expression that was homogenous across the cell population. Plasmids for the various reporter and regulator parts of the circuits are available at Addgene.
Using computational modeling as a guide, the circuit was further adapted for use in mammalian cells. Enhancements to the mammalian gene expression system were sequentially made by identifying additions to the circuit that would improve transcription, translation, and nuclear localization (including addition of an intron, use of a nuclear localization sequence, generation of new TetR-repressible promoters, and more). Gene circuits were successfully developed that showed a linearly tunable expression response to the doxycycline inducer when tested in MCF-7 cells, both for a one- and two-gene linearizer system. The related constructs can be found here.
PCR-mediated Modification of Chromosomal Genes in Yeast
PCR-mediated modification and deletion of chromosomal genes is a tried-and-tested technique for analyzing gene function in S. cerevisiae.
Peter Philippsen's and John Pringle's laboratories have created a set of plasmids that serve as templates for the PCR synthesis of fragments used for a variety of gene modifications. The modifications, based on the groundbreaking work by Baudin et al., include gene deletion, gene overexpression (using the regulatable GAL1 promoter), and tagging with a variety of epitopes and fusion proteins. Browse a table of these plasmids here.
Tim Formosa's lab has constructed 36 plasmids that can also be used to generate linear PCR products. The PCR products can easily be fused to the 3' end of an ORF in the yeast genome, thereby adding a variety of tags preceded by a TEV or PreScission protease site. Browse these plasmids here.
Longtine et al., Yeast. 1998. Jul;14(10):953-61.
March 2013: Fluorescent Biosensors/Markers, Improved Reporter for HTS, New Lentiviral Vectors
New Optogenetic Tool Derived from Box Jellyfish - JellyOp
Optogenetics, a recent technological breakthrough in neuroscience, combines the fields of optics and genetics to allow for precise spatial and temporal control of individual neurons. Photons stimulate cells expressing microbial light-gated ion channels, such as channelrhodopsin-2 and halorhodopsin, to modulate neuronal firing. The laboratory of Robert Lucas designed a new optogenetic tool incorporating an opsin from the box jellyfish to achieve improved sensitivity and reproducibility in signaling. JellyOp is more bleach resistant than existing variants of mammalian rod opsin and allows for sustained signaling under conditions of repeated light exposure. The present JellyOp construct is ideally suited for mimicking the activity of Gs-coupled G protein coupled receptors, while further structural modifications could permit coupling of JellyOp to additional signaling pathways.
Bailes et al., PLoS ONE. 7.(1): e30774. doi:10.1371/journal.pone.0030774.
Additional plasmids for optogenetics research can be found on Addgene's Optogenetics Guide.
Optimized Glutamate-sensing Fluorescent Reporter
Glutamate signaling is important in many species and biosensors are allowing scientists to study this process with greater ease and resolution. The Looger lab has created a new intensity-based glutamate sensing fluorescent reporter and has validated it in a wide variety of neurological systems. To learn more and browse these plasmids, click here.
Marvin et al., Nat Methods. 2013. Feb; 10(2):162-70.
FLuc-P2A-RLuc - A New Reporter System for High Throughput Screening
High throughput screening (HTS) with a single reporter-gene is a convenient method for rapidly assaying diverse compounds (such as small molecules) to identify those that may modulate a specific biomolecular pathway. Reporter-gene HTS identifies the ‘active’ compounds by measuring the amount of the reporter-gene activity compared to controls and thus provides a great starting point for downstream experiments. Unfortunately, direct interactions between compounds and the reporter-gene itself can cause misleading, false-positive results that complicate data interpretation.
To overcome this problem, James Inglese’s lab has devised a “coincidence reporter biocircuit” system that expresses two unique bioluminescent genes (firefly and renilla lucifereases) at equivalent levels in order to efficiently distinguish compounds with active biological activity from those that are interfering with the reporter-gene itself. Because the two luciferase genes are nonhomologous, any compound that shows activity from both reporters has higher probability of being biologically relevant.
Cheng KC, et al. Nat Methods. 2012. Oct;9(10):937.
RGB-Marking with LeGO Vectors
The lab of Boris Fehse initially described their lentiviral "gene ontology" (LeGO) vectors as a system "that allows simultaneous expressing and/or suppression of several genes in a single cell to facilitate the analysis of gene networks." These 3rd-generation lentivectors consist of various combinations of fluorescent markers, promoters/enhancers, and shRNA expression cassettes, all of which can be used in conjunction with each other to visualize complex systems.
The Fehse lab recently published a new application for the LeGO vectors: Red, Green, Blue (RGB) Marking. The fluorescent protein marking system is based on the same principle as TV screens, which combine red, green, and blue beams of light at different intensities to make all colors (see the layman's explanation here). The applications go beyond making pretty images--the ability to produce an almost limitless number of colors allows researchers to identify and track single cell clones. For example, all pink cells in a pink cell cluster are derived from a single pink cell.
This unique marker system gives scientists the means to follow tumor clonalities and to investigate the development of monoclonal or polyclonal metastases. Also, in a setting of organ regeneration after transplantation of stem cells, RGB marking enables the visualization of what individual cells are doing after engraftment. The number of cell clusters with different colors shows the number of engrafted cells, and the size of each cell cluster shows how often the cell has divided after engraftment. There are multiple LeGO vectors available, but all you need to get started with RGB marking is a standard fluorescence microscope and the following three LeGO vectors: LeGO-Cer2, LeGO-C2, and LeGO-V2.
Newest pLX Lentiviral Expression Vectors
From the laboratory of David Root, these newest '300' series pLX vectors (pLEX) are similar in function to the previous 300s, but with new promoters and selectable markers. For example, while pLX301 - 4 utilize the CMV promoter, which is known to be silenced in ES cells, pLEX_307 contains the EF1a promoter which is strongly expressed in ES cells.
The '400' series provides all-in-one doxycycline inducibility. These vectors show almost no leakiness in the off-state, allowing the user to titrate expression by varying the levels of doxycycline. This is ideal for avoiding spurious phenotypes caused by expression far above endogenous levels, such as when performing RNAi rescue experiments or when trying to compare activities of gene variants. Available new pLX plasmids include:
Constitutive Lentiviral Expression
- pLEX_305: SV40-puro; PGK-gateway-no tag
- pLEX_306: SV40-puro; PGK-gateway-V5 tag
- pLEX_307: SV40-puro; EF1a-gateway-V5 tag
Inducible Lentiviral Expression
December 2012: New Tools for Imaging, Golden Gate TALEN Add-Ons, & More
New TALEN Destination Vectors – pc-GOLDYTALEN & RCIscript-GoldyTALEN
Transcription activator-like effector nucleases (TALENs) consist of assembled DNA binding motifs coupled to FokI nuclease monomers that can dimerize and introduce a DNA double strand break. In the past year TALENs have become the tool for genome editing primarily due to their simple and straightforward design and assembly strategies. The laboratory of Dan Carlson and Stephen Ekker designed a new and improved TALEN scaffold, GoldyTALEN, truncated at both the N and C terminus and inducing higher mutation rates than the parental pTAL vector. Dan Carlson deposited 2 destination vectors containing the GoldyTALEN compatible with the Voytas lab Golden Gate TALEN kit. pC-GoldyTALEN directs expression of TALENs from a truncated CAGs promoter. RCIscript-GoldyTALEN is designed for in vitro synthesis of TALEN mRNAs. Both 5’ and 3’ Xenopus β-globin UTRs are included in the vector to enhance expression of the message.
Carlson et al., PNAS. 2012. Oct; 109(43):17382-7.
Improved Genetically Encoded Calcium Indicators - GCaMP6 Variants
The capacity to image and measure neuronal activity in vivo has been improved by the development of various calcium (Ca2+) indicators. Such indicators bind Ca2+ and induce a change in fluorescence signal, allowing scientists to measure action potentials and other receptor activation events which trigger Ca2+ fluxes. Genetically encoded calcium indicators (GECIs), such as GCaMP, express indicators in specific tissues or cells.
Douglas Kim’s lab at Janelia Farm recently developed and deposited novel GCaMP6 variants. pGP-CMV-GCaMP6s, pGP-CMV-GCaMP6m, and pGP-CMV-GCaMP6f have increased ΔF/F0 and faster kinetics compared to previous GCaMP3 and GCaMP5G.
Imaging neuronal calcium responses with novel GCaMP6 sensor variants
(A) GCaMP5G (Akerboom et al., 2012) basal fluorescence in rat neurons transfected in culture. Scale bar: 100 µm. (B) GCaMP6s (manuscript submitted) basal fluorescence. (C) Peak GCaMP5G response to 1 action potential stimulus. (D) Peak GCaMP6s response. Fluorescence change (ΔF/F0) is shown in color. (E) Averaged fluorescence traces of neurons after 1 action potential stimulation (arrow) comparing GCaMP6s, 6m, and 6f with GCaMP3 (Tian et al., 2009) and GCaMP5G. GCaMP6 variants were named based on their slow, medium, and fast response kinetics. Data from Tsai-Wen Chen, Trevor J. Wardill, Eric R. Schreiter, Rex A. Kerr, Vivek Jayaraman, Loren L. Looger, Karel Svoboda, Douglas S. Kim; Genetically-Encoded Neuronal Indicator and Effector Project, Janelia Farm Research Campus, Howard Hughes Medical Institute, www.janelia.org/genie.
Live Visualization of Single mRNAs with MS2 and PP7 Systems
Fluorescent in situ hybridization (FISH) has been considered to be the gold standard for labeling nucleic acids in their native environment; however, the technique lacks spatiotemporal resolution available in living cells. An alternative imaging technique has been developed in the laboratory of Robert Singer that permits live detection of single mRNAs by tagging mRNAs of interest with a repeated MS2- or PP7-derived nucleotide sequence. These binding site sequences form hairpin loops that can subsequently bind to the corresponding MS2 or PP7 coat proteins tagged with a fluorescent protein. Modified MS2 and PP7 coat protein constructs, tdMCP and tdPCP respectively, improve labeling and imaging of target mRNAs by reducing fluorescent background to allow for more intricate investigation of mRNA processing.
Wu et al., Biophys J. 2012. Jun 20;102(12):2936-44.
Enhanced FRET Pairing using Clover and mRuby 2
Förster (or Fluorescence) Resonance Energy Transfer (FRET) is an important tool for determining whether two fluorophores are within a certain distance of each other, and is widely utilized to observe and quantify dynamic biological processes. Historically, CFP and YFP have been the most common FRET fluorophore duo; however, limitations such as emissions overlap between the donor-acceptor pair, sub-optimal FRET efficiency/dynamic range, and low photostability make constructing improved fluorophores desirable.
Michael Lin’s group at Stanford University has recently engineered the novel Clover-mRuby2 FRET pair which shows not only the brightest fluorescence for their respective colors (green and red), but also improves FRET efficiency, dynamic range, and photostability while limiting emissions overlap. The Clover-mRuby2 couple was tested in 4 established FRET reporters (Camuiα-CR, AKAR2-CR, VSFP-CR, and Raichu-RhoA-CR) with noticeable improvement, making these updated sensors more useful in detecting rapid cellular processes in real-time. This new FRET pair has great potential not only for enhancing existing sensors, but also for the construction of new, more sensitive FRET reporters.
Lam et al., Nat Methods. 2012. Oct; 9(10):1005-12.
A New Split GFP for Studying in vivo Protein-Protein Interaction – spGFP
A new superpositive split GFP (spGFP) construct for detecting protein-protein interactions in vivo at room temperature was develop by Brian McNaughton's lab. The superpositive split GFP has a greater reassembly speed than previous split GFP constructs. The increased positive charge significantly reduces protein aggregates. This new design also shows robust signal at room temperature making this ideal for studying protein-protein interactions in vivo. The pET11a-Z-NspGFP and pMRBad-Z-CspGFP plasmids are available through Addgene.
Blakeley et al., Mol Biosyst. 2012. Aug; 8(8):2036-40.
EM Imaging in all Cellular Compartments Using APEX
A key component of creating high-resolution electron microscopy (EM) images is contrast. Existing genetic tags designed to increase EM contrast, such as horseradish peroxidase (HRP), are helpful in some cellular locales, but have restrictions. In their recent Nature Biotechnology article, the MIT laboratory of Alice Ting describes the engineering of ascorbate peroxidase (APX) to make a new genetic tag that overcomes the shortcomings of previous EM reporters. The Ting lab introduced a series of mutations to APX to change it from monomeric to dimeric, as well as more highly active towards DAB, thus creating enhanced APX (APEX).
APEX offers the following advantages as a genetic tag EM reporter:
- Fixation and staining of cells does not require a detergent, allowing ultrastructure to be maintained
- APEX does not require light, is easy to use and should have applicability to tissue samples
- The nature of APEX staining makes it a useful tool for 3-D EM applications
- APEX can be fused to fluorescent proteins, allowing for correlative studies using both light microscopy and EM
- The DAB stain generated by APEX is tightly localized, giving spatial resolution in EM on the order of 10 nm
- APEX works in all cell compartments tested, including the cytosol, nucleus, mitochondria, and endoplasmic reticulum
COS7 cell in the metaphase of mitosis
The chromosomes are aligned at the center of the cell. The cell is expressing APEX-Histone2B, which causes APEX to be incorporated throughout chromatin structures. This cell was fixed and stained with 3,3'-diaminobenzidine, resulting in a dark reaction product that highlights chromatin and is visible under a conventional light microscope (top left). The cell was subsequently processed for electron microscopy and imaged with much higher resolution under the electron microscope (low magnification EM at top right; high magnification EM at bottom). Image courtesy of the Ting lab.
APEX plasmids are now available at Addgene.
Martell et al., Nat Biotechnol. 2012. Nov; 30(11):1143-8.
Improved Mos-1-mediated Transgenesis Reagents for C. elegans
Erik Jorgensen's lab had previously created a set of plasmids for targeted transgene insertions (Mos1-mediated single-copy transgene insertions; MosSCI) and targeted deletions (Mos1-mediated deletions; MosDEL) in C. Elegans. In their 2012 Nature Methods paper, they add to this collection, making it even easier to use.
First, they have created a Mos-1 expression vector under the eft-3 promoter that significantly increases the insertion and deletion frequencies. Second, they introduce a set of plasmids to facilitate selection of transgenic strains with antibiotic markers and additional transgene insertion sites. Plasmids from the paper are available from Addgene.
Frokjaer-Jensen et al., Nat Methods. 2012.; 9(2):117-8.
September 2012: Add-Ons for the Golden Gate TALEN Kit, Brighter ECFPs, and More
A Brighter ECFP Variant – mTurquoise2
Cyan based fluorescent proteins suffer from low quantum yield and hence are mostly used as the acceptor, rather than donor, in FRET assays. Using site-directed mutagenesis and fluorescent lifetime-based screening, the Dorus Gadella lab identified mTurquoise2, a variant of ECFP (enhanced cyan fluorescent protein). mTurquoise2 has the highest quantum yield of any monomeric fluorescent protein. In vivo studies in mammalian cells show a 20% gain of brightness, high photostability and better performance in FRET studies.
Addgene distributes several mTurquoise2 vectors with various targeting sequences - mitochondria, nucleus, ER, etc.
Goedhart et al., Nat Commun. 2012.; 3: 751.
Drosophila phiC31 Transgenesis Vectors – pBID and pMartini Gate
Advanced genetic tools available for examining gene function in Drosophila have strongly contributed to its widespread use as a model organism in the genomics era. Integration of transgenes into the Drosophila genome via phiC31 integrase permits efficient and site-specific targeting.
The laboratory of Brian McCabe at Columbia University has recently improved upon phiC31 transgenesis by creating a series of vectors for expressing transgenes in Drosophila. The pBID series of plasmids incorporates several design enhancements, including: (1) gypsy insulator sequences to permit uniform expression levels independent of genomic integration site; (2) backwards compatibility with pUAST cloning sites; (3) Gateway cloning compatibility; (4) DSCP promoter with 10 UAS binding sites to improve expression while eliminating leaky expression in the absence of GAL4; and (5) a variety of fluorescent protein or epitope tagged constructs. The pMartini-Gateway series of plasmids, a set of intermediate vectors, was generated concurrently with the pBID series to assist in Gateway cloning and in developing novel destination vectors.
Wang et al., PLoS ONE. 2012.; 7(7): e42102.
Drosophila transcription factor ORFs
Drosophila melanogaster, one of the best-known model organisms, has been used to advance our knowledge of genetics and developmental biology since first employed over 100 years ago. The complete sequence of the fly genome, published back in 2000, opened many doors to investigating not only the 15,000+ genes, but also the >60% of functional, non-coding DNA found within it. Over the course of the last decade, many methodologies have been employed to identify some of the regulatory elements found within the non-coding DNA; however, the specific functions of these have not been deeply explored as this requires not only identifying the elements, but also the transcription factors that bind to them.
Bart Deplancke’s group from the Laboratory of Systems Biology and Genetics at the Swiss Federal Institute of Technology has recently deposited a nearly complete collection of Drosophila transcription factor open reading frames (ORFs) comprised of 692 plasmids. These ORFs were cloned open-ended into Gateway compatible Entry vectors, permitting easy and efficient subcloning for a variety of downstream applications. Using this clone library, the authors developed and validated a gene-centered, high-throughput yeast 1-hybrid system by which they identified some previously uncharacterized direct interactions between transcription factors and Drosophila cis-regulatory elements. This ORF library will be a significant resource for many scientists studying the biological importance of specific DNA-protein interactions within the Drosophila regulatory gene network.
Browse this whole collection of Drosophila transcription factor ORFs available through Addgene.
Hens et al., Nat Methods. 2011.; 8(12): 1065-70.
Add-Ons for the Golden Gate TALEN Kit
The Golden Gate TALEN kit, deposited by the Voytas and Bogdanove labs, has proven to be Addgene's most popular kit. Due to its popularity, a number of labs designed new plasmids to be used in conjunction with this powerful tool. In the summer of 2012, three new Addgene depositors contributed destination vectors compatible with this kit:
pCS2TAL3-DD and pCS2TAL3-RR
Developed in the lab of David Grunwald, pCS2TAL3-DD and pCS2TAL3-RR are next generation TALEN backbone vectors in place of pTAL1, 2, 3, or 4 from the Golden Gate TALEN kit. For both plasmids, sequence positions 1214–2210 of pTAL3 were cloned into a pCS2 expression vector resulting in shorter N- and C-terminal tal protein segments (136AA and 63AA, respectfully). This next generation architecture has been shown to increase mutation induction when using TALENs. The FokI domains (DD, RR) used are obligate heterodimers that require cloning of left and right TALEN monomer proteins into opposite vectors.
Dahlem et al., PLoS Genet. 2012.; 8(8): e1002861.
pTAL5-BB and pTAL6-BB
Plasmids pTAL5-BB and pTAL6-BB were created in Tom Ellis’ lab and function as alternative destination vectors to generate TAL Orthongal Repressors (TALORs). TALORs can be used to custom repress gene expression in yeast. They consist of the DNA-binding domain of a TALE and strong nuclear localization tags. TALORs repress transcription initiation when targeted to DNA sequences within core promoter regions. pTAL5-BB contains the GAL1 promoter, placing TALORs built into this vector under galactose-inducible expression. pTAL6-BB contains the TEF1 promoter, resulting in constitutive expression of TALORs built into this vector.
Blount et al., PLoS One. 2012.; 7(3): e33279.
pCAG-T7-TALEN(Sangamo)-Destination with homo- and heterodimeric FokI domains
pCAG-T7-TALEN(Sangamo)-Destination constructs were designed by Pawel Pelczar’s lab for the purpose of optimal mammalian expression of Voytas Golden Gate-assembled TALENs, both in microinjected embryos and transfected cells. TALEN expression is driven by the strong CAG promoter or can be achieved by in vitro mRNA synthesis from the T7 promoter. Truncations were introduced to the N- and C-terminus of the pTAL3 TALEN backbone, which were initially published by Sangamo BioSciences (N153AA, C63AA) and showed robust cleavage activity in several later studies. pCAG-T7-TALEN(Sangamo)-Destination vectors are available with homodimeric or enhanced heterodimeric (ELD, KKR mutations) FokI domains.
June 2012: Promiscuous Biotin Ligases, QB3 MacroLabs Expression Vectors, GCaMP5, and more
Promiscuous Biotin Ligases
In recent years there has been a significant focus on finding a protein’s “interactome”, identifying neighboring and potentially interacting partners of your protein of interest. Current means of identifying one’s network are limited and have several drawbacks, for example biochemical approaches encounter problems with insolubility of expressed proteins and in Y2H systems the interactions are tested outside of the natural environment.
In his recent paper, Kyle Roux developed a simply and quick technique for identifying interacting proteins. The system relies on promiscuous biotin protein ligase fused to a protein of interest; once the culture media is supplemented with biotin, the ligase will biotinylate proteins that are in close proximity. Biotinylated proteins can then be captured by affinity purification and identified using mass spec.
Specifically, Roux et al utilize a mutated form of the E. coli DNA-binding biotin protein ligase, BirA. BirA* (with R118G mutation) lacks the specificity of BirA and has been shown to promiscuously biotinylate proteins in a proximity dependent fashion. Roux et al test their system on lamin-A, a structural element of the nuclear envelope, thus identifying proteins that interact and/or are in close proximity to lamin-A.
Roux et al., J Cell Biol. 2012. Mar 19;196(6):801-10. Epub 2012 Mar 12.
Expression Vectors from the QB3 MacroLab
Finding the right empty vector for your purification technique of choice is often challenging. Finding a vector with the right tags and the right fluorescent fusion protein is even more challenging. What are the chances that someone has a backbone with just the right combination of tags and fusion proteins?
Actually, if you look at Addgene's collection of backbones from QB3 MacroLab, your chances are pretty good. This core facility for the California Institute for Quantitative Biosciences (QB3) designed these backbones to offer different combinations of the following TEV-cleavable N-terminal tags: His6, MBP, FLAG, NusA, Mocr, proteingG, StrepII, Sumo, gCrystallin, N10, and Biotin. The N-terminal tags can be found in various combinations with the C-terminal fusions that include mCherry, mOrange, mCitrine, msfGFP, and mCerulean. Most of the vectors were designed for bacterial expression, but there are also baculovirus and mammalian expression backbones.
The vectors were designed for ligation independent cloning (LIC), for which there are convenient protocols on the MacroLab website. You can also download a full expression vector list in Excel format from their website.
GCaMP5: Out With the Old and In With the New
The very popular calcium sensor GCaMP3 has been updated. GCaMP5G, a.k.a. GCaMP3-T302L R303P D380Y, has improved deltaF/F0 and lower F0 as compared to GCaMP3. Addgene now offers pCMV-GCaMP5G from Loren Looger's lab or the membrane targeted version, Lck-GCaMP5G, from Baljit Khakh's lab.
Zinc Finger Arrays Targeting Endogenous Zebrafish Genes
Gene targeting is a powerful technique that induces specific DNA stand breaks followed by stand rejoining to modify genes of interest. Engineered zinc-finger nucleases (ZFNs) are widely used for targeted genome modification in Drosophila, C. elegans, D. rerio, plants and humans. ZFNs function as dimmers and consist of a DNA-binding zinc finger domain that is covalently linked to a non-specific DNA cleavage domain of a restriction endonuclease, such as FokI. When the two zinc finger DNA binding domains bind to the target sites, the cleavage domains are able to dimerize and cleave at the desired target site. In order to provide researchers with easy-to-use zinc finger arrays, the laboratories of Keith Joung, Randall Peterson and Joanna Yeh at Massachusetts General Hospital have produced 49 pairs of zinc finger arrays targeting a variety of zebrafish genes.
Methods for engineering zinc finger domains:
New Retroviral Vectors for Cellular Reprogramming
Alzheimer’s disease is a neurodegenerative disorder for which there is no known cure. The disease most often arises sporadically, but familial forms can also be genetically inherited. The majority of research to date has focused on the less prevalent familial form, as this is more easily modeled in the lab. A new technology used to reprogram primary cells into induced pluripotent stem cells (iPSCs) has been successfully utilized in the study of some neurological diseases; however, it is not known whether this approach would be effective for studying both the sporadic and familial forms Alzheimer’s disease. Steven Dowdy and Larry Goldstein at UCSD have developed retroviral plasmids to generate iPSCs from the primary cells of Alzheimer’s patients. The resulting data provides evidence that iPSCs are effective for studying pathogenesis at the early stages of sporadic and familial Alzheimer’s disease, and can be successfully used in patient-specific cells. This technology not only provides a means to study the mechanisms of disease pathogenesis, but may also prove to be a useful tool for Alzheimer’s disease diagnosis.
Israel et al., Nature. 2012. Jan 25;482(7384):216-20.
March 2012: O-phosphoserine, Genome Wide Knockdown, Brain MiniPromoters, and Cyclin Reporters
Engineering Phosphoproteins in E. Coli
Protein kinases post-translationally phosphorylate specific residues as a means of regulating cell-signaling cascades. O-phosphoserine (Sep) being, by far, the most common phosphorylation modification. Phosphorylation events often require specific stimuli or conditions that are difficult to experimentally replicate, limiting investigations into the functional roles of these phosphoamino acids. Jesse Rinehart’s group at Yale has devised a system to incorporate Sep directly into the genetic code of E. coli using a re-engineered tRNA (tRNAsep). This process requires not only the orthogonal tRNAsep:Sep-tRNA synthetase pair, but also a modified EF-Tu, which allows tRNAsep to be incorporated during protein synthesis. This study elegantly deduces the minimal requirements for genetic code expansion as well as provides a unique tool for protein engineering and research.
Park et al., Science. 2011. Aug 26;333(6046):1151-4.
See details on the Rinehart & Söll Phosphoprotein Synthesis Kit here.
Genome-wide shRNA Knockdown Screens Made Easier with DECIPHER
In 2011, Addgene began collaborating with Dr. Alex Chenchik of Cellecta, Inc. and Dr. Gus Frangou of the Fred Hutchinson Cancer Research Center to distribute Cellecta's DECIPHER pooled lentiviral shRNA libraries. The DECIPHER Project was designed to make high-throughput RNAi genetic screening accessible to academia.
Each pooled shRNA library (or module) targets approximately 5,000 genes/transcripts, with 5 to 6 bar-coded shRNAs per target gene to maximize screening efficiency. The library modules were constructed as a series with non-overlapping sets of target genes. There are currently three modules for human and two modules for mouse available which target 15,000 and 10,000 genes, respectively. The first two library modules for human and mouse target mostly well characterized pathway-associated and disease-associated genes. The third human module targets cell surface and DNA-binding proteins, and other conserved genes. Just like Cellecta's custom libraries, the DECIPHER Project libraries are constructed with shRNA expression cassette oligonucleotides containing unique sequence tags ("bar-codes") synthesized on Agilent's array-based platform. Cellecta also offers free bar-code analyzer/deconvoluter software to assist scientists in processing Illumina HT Sequencing data generated from their RNAi screens. For more information about this powerful new screening tool, visit http://www.addgene.org/decipher/.
MiniPromoters for Brain-Specific Gene Expression
Region and cell-specific expression of a gene of interest is a critical experimental consideration, especially when studying the human brain which by some estimates contains 100 billion cells and possibly 100 trillion cellular connections. In order to address the lack of tools available for brain-region specific gene expression, a team of investigators joined forces to create the Pleiades Promoter Project (www.pleiades.org). Using a novel design pipeline, they produced MiniPromoters derived from endogenous human gene promoter sequences and combined them with reporter genes such as EGFP, EGFP/Cre or LacZ in constructs designed for knock-in insertion of transgenes to yield region specific expression as verified by in vivo experiments in mouse brain. Some MiniPromoters produced neuron or glia specific expression, as expected based on the gene the promoter was taken from, while others produced interesting, but unrelated expression patterns. The MiniPromoters available in the Pleiades Plasmids offer researches a more refined tool to obtain localized gene expression in the brain than previously available.
Portales-Casamar et al., Proc Natl Acad Sci USA . 2010. Sep 21; 107(38):16589-94.
Cyclin D1 Reporters
Regulated progression through the cell cycle requires sequential expression of a family of proteins called cyclins. Frank McCormick's group at UCSF recently deposited a series of human cyclin D1 promoter constructs from his Nature 1999 publication with Addgene. Cyclin D1 is over-expressed in many colon carcinomas and has been identified as a target of β-catenin mediated transcription via the core TCF/LEF-binding consensus sites in the promoter region. The McCormick group demonstrates this regulation using Cyclin D1 luciferase reporter constructs containing mutated TCF-binding sites.
Tetsu O, McCormick F. Nature. 1999l. Apr 1;398(6726):422-6.
December 2011: GECOs, iMNs, Zinc Fingers, and Multicistronic Drosophila Vectors
New Flavors for Genetically Encoded Ca2+ Indicators (GECOs)
Fluorescent indicators have long been used to measure intracellular Ca2+ levels, an important process in many signaling activities. In a recent Science paper, Robert Campbell’s laboratory at the University of Alberta describe an improved screening method for detecting changes in Ca2+ dependent fluorescence that they used to develop improved genetically encoded Ca2+ indicators, dubbed GECOs. The green (G-GECOs) demonstrate a two-fold improvement in Ca2+-dependent change in fluorescence over earlier fluorescent protein Ca2+ indicators. Blue-shifted (B-GECO) and red-shifted (R-GECO) Ca2+ indicators fill a void in the spectrum of available colors for genetically encoded Ca2+ indicators. By recombining these newly created constructs, additional ratiometric GECOs, GEM-GECO and GEX-GECO, were developed. The GECO plasmids offer researchers additional choice and flexibility when selecting fluorescent Ca2+ indicators.
Zhao et al., Science. 2011. Sep 30;333(6051):1888-91.
Transcription Factor Mediated Reprogramming of Fibroblasts into Motor Neurons (iMNs)
Studying cellular subtypes of the central nervous system (CNS) has proven to be challenging. For one, isolating subtypes of the human CNS can be strategically limiting in terms of supply and attainability. Moreover, differentiating embryonic stem cells (ESCs) into neuronal sub-populations has been fairly unsuccessful to date. Kevin Eggan's group at Harvard recently showed that they could use transcription factor-mediated expression to induce mouse and human fibroblasts into spinal motor neurons. This particular subset of neurons controls the contraction of muscle fibers involved in movement. Characterization of these reprogrammed cells showed they exhibit a distinct motor neuron identity . The complete set of reprogramming transcription factors are available at Addgene cloned into a retroviral backbone.
Son EY et al., Cell Stem Cell. 2011. Sep 2;9(3):205-18.
Zinc Finger Nuclease Assembly in Zebrafish
Zinc Finger Nucleases (ZFNs), chimeric fusions between a zinc-finger protein and the nuclease domain of FokI, are used in a wide range of model organisms for gene inactivation. Gene disruption occurs by imprecise repair of a ZFN-induced double-strand break within the coding sequence of a target gene. Using the modular assembly-based approach to ZFNs construction, the Lawson and Wolfe labs from UMass Worcester created a library of over 70 different zinc-finger cassettes. In parallel, the labs assembled a new publicly available database of zebrafish genes that can be targeted by ZFNs. The Lawson and Wolfe labs also demonstrate that ZFNs are a viable tool for creating an heritable gene inactivation in vertebrates by creating several germline mutations in zebrafish.
Zhu et al., Development. 2011. Oct;138(20):4555-64.
Vectors for Simplified Multicistronic Expression in Drosophila
Techniques to produce stable mammalian cell lines have been around for years; however, the ability to stably transform insect cells is especially limited. Because of these limitations, cell-culture based approaches are generally disregarded when attempting to validate chemical/RNAi library screens or produce recombinant protein. The Sutherland Lab has developed two Droshophila vectors, pAc5-STABLE1-Neo and pAc5-STABLE2-Neo, which facilitate the multicistronic expression of proteins without relying on co-transfection with a second vector (for antibiotic selection) or the need for dual-promoters. Both of these versatile vectors allow for either N- or C-terminal tagging with a fluorescent protein and pAc5-STABLE2-Neo gives the additional option of co-expressing a separate fluorescent tag, that can by used for FACS analysis.
Gonzalez et al., Scientific Reports. 2011. Aug; 1, Article: 75.
September 2011: TALENS, Photoswitchable Proteins, a Novel iPSC Factor and More
PSmOrange- a Novel Photoswitchable Protein
Photoconvertible proteins are widely used in tracking the migration, fate, and dynamics of cells, organelles, and proteins. Photoconvertible proteins can be divided into those that are photoactivatable- that can be turned on or off; or those that are photoswitchable- that can switch from one color to another. In a recent Nature Methods publication by Vladislav Verkhusha’s group at Albert Einstein, a novel photoswitchable protein, PSmOrange was characterized. PSmOrange is initially Orange (excitation, 548 nm; emission, 565 nm) but becomes far-red (excitation, 636 nm; emission, 662 nm) after irradiation with blue-green light. The photoconverted or the far-red version of PSmOrange is brighter than conventional far-red fluorescent proteins and can be imaged in deep tissues. PSmOrange is in an easy to clone vector and has been used to generate numerous fusion proteins.
Subach et al., Nat Methods. 2011. Jul 31;8(9):771-7.
Golden Gate TALEN and TAL Effector Kit
TAL effector nucleases (TALENs) are fusion proteins containing transcription activator-like (TAL) DNA binding domains fused to the FokI nuclease. TALENs have shown to be robust tools for DNA targeting in a variety of species. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. The Voytas laboratory at the University of Minnesota recently published a system for custom TALEN design and assembly. The Golden Gate TALEN and TAL Effector Kit and accompanying documentation allow one to efficiently assemble TALEN constructs with custom repeat arrays, containing anywhere between 12 and 31 of these repeats. The reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest.
Cermac et al., Nucleic Acids Res. 2011. Jul;39(12):e82.
GLI Transcription Factor Improves iPSC Generation
Generating induced pluripotent stem cells (iPSCs) from somatic cells involves finding the “right” combination of factors. The transcription factor Myc has been used successfully in producing iPSCs (in combination with other proteins)- although these mice have been shown to develop cancer. The Yamanaka lab at Kyoto University recently performed a screen to find a transcription factor that could improve efficiency of iPSC generation and potentially improve the viability of iPSC mice. Glis1 (Glis family zinc finger 1) was shown to promote iPSC generation in combination with Oct3/4, Sox2, and Klf4 in both mouse and human fibroblasts- replacing the requirement of Myc in generating iPSCs. Since the beginning of the summer, Glis1 has been requested by over 60 laboratories and continues to be a gene of interest for many researchers. Additional reprogramming factors are also available at Addgene.
Maekawa et al., Nature. 2011. Jun 8;474(7350):225-9.
New Recombinases for Animal Genomes
Site-specific recombination is a robust tool used for controlling gene expression. Site-specific recombinases can be used in animal models to turn expression of a gene on or off, create conditional mutants, or introduce novel genetic material. Gerald Rubin’s lab at the Janelia Farm Research Campus has characterized a set of novel recombinases from yeast- KD, B2, B3, and R. All recombinases were shown to be active in Drosophila and do not cross-react. In addition, KD and B3 were shown to be non-toxic and active in mice. Increasing the number of functional, specific recombinases allows for greater flexibility when performing experiments that involve multiple recombination events.
Nern et al., Proc Natl Acad Sci U S A. 2011. Aug 23;108(34):14198-203.
RNAi vectors for improved detection and inducible expression
To improve the efficiency of shRNA delivery and expression, specifically in targeting genes involved in proliferation and survival, Scott Lowe’s laboratory at Cold Spring Harbor has designed a set of plasmids that can be used for tracking and induction of retroviral-mediated shRNA expression. The retroviral plasmids express two transcripts. The first transcript, driven by a tetracycline response element (TRE)-encodes for a dsRed tagged miR-embedded shRNA, while the second transcript encodes for Venus and a mammalian antibiotic resistance cassette or Tet-inducible transactivator (rtTA3). The dual fluorescent system adds sensitivity to detection and the TRE allows for targeted knockdown, helping mimic in vivo therapeutic strategies. The TRMPV vectors, as they’re called, come in a variety of flavors- with different antibiotic resistance cassettes- and make up part of Addgene’s collection of inducible shRNA plasmids.
Zuber et al., Nat Biotechnol. 2011. Jan;29(1):79-83.
STAG2 and Aneuploidy
Todd Waldman’s lab at Georgetown recently showed that mutations in STAG2 can result in aneuploidy. Aneuploidy-- an abnormal number of chromosomes-- is often seen in cancer cells. STAG2 is a member of the cohesin complex, which regulates the separation of sister chromatids during cell division. In the Waldman lab’s recent Science paper (Solomon et al., 2011)- they identify specific STAG2 mutations found in cancer cells- that result in chromosomal disruption. STAG2 plasmids, wild-type and these aneuploidy-associated mutant forms, are now available through Addgene; along with a set of shRNAs targeting STAG2.
Solomon et al., Science. 2011. Aug 19;333(6045):1039-43.
Hot Article Contributors:
Addgene's Hot Article page is a compilation of summaries describing new plasmids available at Addgene and their related journal articles. These summaries were written by: Andy Baltus, Melina Fan, Matthew Ferenc, Benoit Giquel, Melanie Herscovitch, Caroline LaManna, Jason Niehaus, Marcy Patrick, Eric Perkins, Brook Pyhtila, Lianna Swanson, and Julian Taylor-Parker.