...various cyan fluorescent proteins is depicted in figure 1. Of note, the photobleaching rate does not depend ...to be used with the fluorescent protein. In figure 1 we provide an example of such a comparison between...the variants is comparable (lower panel of figure 1). To avoid changes in excitation power, it is important...different ways. We describe a number of issues with some of the experimental designs used to make these...of Amsterdam. The previous post in this series described a practical approach to selecting a bright fluorescent... protein. Photobleaching is the irreversible destruction of a fluorophore under the influence of light...photobleach at some point. For live-cell imaging, it is desirable to have fluorescent proteins that are photostable...

...Cas9 can be used to modify any desired genomic target provided that (1) the sequence is unique compared...will cover three ways to circumvent this limitation: 1) the use of novel S. pyogenes Cas9 variants with varying...Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase smaller than SpCas9, so it can be packaged...Biol 19: . https://doi.org/10.1186/s13059-018-1591-1 Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, ... may be unintentionally mutated along with the desired target locus. The PAM sequence is of particular...

...neuron which generates an action potential (Figure 1). This light-induced excitation can take place in ...channelrhodopsin activation to a small volume of brain tissue (~1 mm3). Current channelrhodopsins also need high-intensity...fiber-optic cables into the brain. Figure 1: With no blue light, the channelrhodopsin remains ... model to help them construct “designer” channelrhodopsins with desired characteristics. The model was...Viviana Gradinaru’s lab used machine learning to design new channelrhodopsins that are more light sensitive...constrained and poorly understood. It’s hard to design new channelrhodopsins with high-throughput proteins...Using machine-learning to overcome channelrhodopsin design challenges To overcome these challenges, the Arnold...

... is conjugated to a fluorophore (Figure 1).   Figure 1: Comparison between how direct immunofluorescence...important considerations are to make sure your secondary 1) will recognize the species of your primary antibody...New York, pp 299–311. https://doi.org/10.1007/978-1-4939-8935-5_26 Stuurman N. (2012) Introduction to ...antibody-based technique, validation and proper experimental design are essential. In this article, we’ll go through...steps include: counterstaining, mounting sample on slides, and confocal   Figure 2: High-level overview...

...standard for genetically-encoded FRET donors (Fig. 1). Scores of FP variants have been created, thereby...with finesse using tRNA suppression technology (Fig. 1) (Dumas, Lercher, Spicer, & Davis, 2015). By using...orthogonal labeling strategies are presented (Fig. 1) which can be carried out using Addgene plasmids. ...involved in skeletal muscle contraction.   References 1. Degorce, François, et al. "HTRF: a technology tailored... transfer measurements of ryanodine receptor type 1 structure using a novel site-specific labeling method...package. One important concept in FRET experimental design is that the targeting specificity requirements ...codon (often amber i.e. UAG) engineered into a desired site. During translation, the novel tRNA/unnatural...

..., including those found at Addgene! Figure 1: Strategy for assembling the barcoded gRNA library...pool-clone them into the pAWp28 storage vector (Figure 1): Forward oligo: 5’- CACCGNNNNNNNNNNNNNNNNNNNGTTTGGGTCTTCGAGAAGACCTATTCXXXXXXXXC... He can be contacted at [email protected].   References 1. Cheng, Allen A., Huiming Ding, and Timothy K. Lu.... Blog Learn Best Practices for Designing Your gRNA Read How Barcodes Can Be Used in Lineage Tracing Read...genetic perturbations. The ease with which one can design and synthesize CRISPR guide RNAs (gRNAs) for genome...their functions. The continual advancements in gRNA design necessary to achieve maximal on-target and minimal...regulation in biological systems. The CombiGEM platform provides a means to create barcoded gRNA libraries that...

...unleashes its cargo – its own genome.   Fig. 1: Viral particle infection into a host cell. Image ...Vector Resources at Addgene! References and Resources 1. Roe T, et al. “Integration of murine leukemia virus...nuclear import of human immunodeficiency virus type 1 preintegration complexes.” Proc. Natl. Acad. Sci. ...target cells and organisms, and when that cargo is designed to make you sick…well, you feel it. But the same...host genome or temporarily express whatever it encodes. This ability to infect cells with a high efficiency...

...were found on the plasma membrane (top panel in Fig. 1). Of course, this was problematic, because the DREADD...plasma membranes in both species (bottom panel in Fig. 1). Our results indicate that the tag protein may have...protein of interest the membrane.   Figure 1: Electron microscope images showing ultrastructural...protein-coupled receptor, one of the Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) (Armbruster...bottom). The images show examples of dendrites (blue shades) in the monkey amygdala. Red and green arrows point...deciding on the appropriate tag, the experimental design should be the main guidance. The results from our...Raper J, Hu X, Paré J, Bonaventura J, Richie CT, Michaelides M, Mueller SAL, Roseboom PH, Oler JA, Kalin NH...

...Transfer Vectors   Expressing your gene of interest pMKO.1 GFP (Plasmid #10676) Retroviral vector for cloning...GFP expression.  Cloning and expressing RNAi pMKO.1 puro (Plasmid #8452) RNA interference (RNAi) is an...information and a detailed protocol on how to use the pLKO.1 vector can be found here. Cre-Lox technology Puro....of the Retroviridae or Retrovirus family, which includes the gamma-retrovirus and lentivirus genera, have...dividing and non-dividing cells. More detailed descriptions of the lentiviral elements and the 2nd and 3rd...#48201) This retroviral vector from the Gage lab encodes GFP and Cre-recombinase. The Cre-lox system is ...building block Entry vectors and 5 lentiviral Destination vectors. These vectors can induce combinatorial...

...overlap with the negative control population (Figure 1). Based on where the GFP- population ends, you can...total cells present in the gate.   Figure 1: Example of yes-no flow plot of the (negative) control...for a yes-no dot plot, like the one shown in Figure 1.  Figure 3: An example of a histogram plot ... that you know how to read flow plots and have designed your first flow panel, you’ll load your samples...

...succumb to the toxicity of CcdB.   Figure 1: Image from Bernard, P., et al. showing the pKIL 18...submit a guest blog entry of your own! References 1. Bernard, P. “Positive Selection of Recombinant DNA... plasmid ccdB killer gene”. Gene. 1994 Oct 11;148(1):71-4. PubMed PMID: 7926841. 4. Bernard, P., et al... plasmid maintenance during cell division. ccdB codes for the toxic protein (CcdB) that acts as a DNA ...death. ccdA, another gene found in the ccd operon, codes for the antitoxin protein (CcdA) that protects the...of ccdB, leading to positive identification of desired clones.   ...Becomes a powerful cloning tool... efficiently, as those that do not contain the desired insert should not grow.   Figure 2: Image...

...interactions with other peptides, like an Fc-binding domain (Figure 1).  Figure 1: Examples of sites...sites on antibodies used for conjugation. 1) Endogenous lysine residues, which can be found throughout the...we would be remiss not to at least mention. Oligonucleotides allow for sensitive detection of low abundance...Strong protein-protein interactions with other peptides. Created with BioRender.com.     Many biologists... conjugation. But hopefully, this introduction provides a good base from which to dive deeper!References...

...separation is maintained by 2 sets of interactions : 1. Interactions between the non-target strand and a ...theoretically decrease off-target cutting. Figure 1: Strand separation and target DNA binding to the Cas9... improvements in genome engineering! References 1. Slaymaker, Ian M., et al. "Rationally engineered ... nuclease activity of the Cas9 nuclease itself. Design and testing of eSpCas9 Looking at the structure...and the negatively charged DNA and therefore destabilize basal strand separation. This decreased strand... choice and get improved on-target specificity. Design and testing of SpCas9-HF1 Kleinstiver et al and...Joining Repairs Double Strand Breaks Get Tips on Designing Your gRNA Browse Our CRISPR 101 Series Resources...

...vector (abR, Figure 1). The resolution step (“double crossover”) which leads to the desired mutation and excision... requires selection. These two steps are (Figure 1): The initial plasmid integration step (the so-called... loss of a specific phenotype (using sacB, Figure 1, further discussed below).  Counter-selectable marker...Current protocols in molecular biology 106.1 (2014): 1-16. PubMed PMID: 24733238. Additional resources on...scarless mutations, ones that don’t leave any undesired scars in the chromosome. These methods roughly... recombination enzymes can also accomplish the desired genomic modification. This technique is frequently...the pDS132 MCS with a versatile, synthetic MCS (designed with the help of the DNA tuner program) (Latynski...

...the other (Sandomenico et al., 2020). Figure 1: Comparison between the IgG antibody and scFv. ...intensity of a tagged protein. SunTag has two components: 1) a protein of interest that’s tagged with 10-24 copies...Application. Clinical and Developmental Immunology 2012:1–15 . https://doi.org/10.1155/2012/980250  Bates A,...without disulfide bonds, made by molecular evolution 1 1Edited by I. A. Wilson. Journal of Molecular Biology...recombinant antibody. They are ~25 kDa single polypeptides that contain the variable light chain (VL) and...lethal to animals.  In the clinic, scFvs’ size also provides advantages over antibodies (Ahmed et al., 2012... bond between the VL and VH domains. This bond provides stability and solubility for the scFv (Gaciarz...

...difficult-to-manipulate primary cells, achieving knockdown within 1-2 hr of reagent delivery. TRIM-Away is suitable for... inducer levels. That is, if transcription factor 1 controls expression of transcription factor 2, which...modulating the induction of transcription factors 1 and 2. See Aranda-Díaz et al. for more information...TRIM21 recruits ubiquitin and the proteasome to destroy antibody-bound pathogens. Clift et al. found that...necessary for studying endosomes, the Hell lab designed their super-resolution pH indicators (SRpHi) biosensors...sgRNAs are used with FIRE-Cas9, experiments can be designed to target multiple loci at once. In addition, ...

... employing HDR. Ma et al. moved forward with gRNA 1 to conduct microinjection experiments in S phase zygotes...instead from the mother’s wild type allele (Figure 1). 72.4% of embryos carried two wild-type copies of...proceeded through NHEJ rather than HDR. Figure 1: Homology-directed repair in targeted zygotes proceeds...donated skin, blood and semen samples. Ma et al. designed plasmids containing Cas9 and two different gRNAs... at 3 days post fertilization. Since Ma et al. designed a repair template with two synonymous mutations...thus quieting some fears about the creation of “designer babies.” The differences in editing between iPS...

...want a low affinity calcium biosensor, so that the (1) binding of calcium, (2) allosteric rearrangements... to a red fluorescent state only by simultaneous (1) user-controlled illumination with violet light and...fluorescent indicators. Nature protocols. 2006 Aug;1(3):1057-65. Resources on Addgene.org Addgene’s AAV... There are several calcium reporters specially designed for the aims and requirements of in vivo studies...populations of interest. Many of the calcium sensors described are also already packaged into viral vectors and... L, Campbell RE. Structure-and mechanism-guided design of single fluorescent protein-based biosensors....

... : Niroshi and Ben describe their projects 0:28 - 1:12 : Introduction from Tyler 1:13 - 2:02 : Niroshi...

... the Roska lab used four different design strategies: Strategy #1 (ProA): Create promoters with sequences...cell types in mice. Figure 1: The four strategies used to design synthetic promoters. Legend: TSS...rate of targeted expression. Surprisingly, <1% of synthetic promoters replicated the expression specificity...expression in the retina.  Four approaches for designing synthetic promoters  To build a library of synthetic...promoters with the help of epigenetic analysis and designed using stretches of DNA that were transcriptionally...class of cells  Of the four different promoter designs, promoters from strategy #4 had the highest rate...here.  Applications for synthetic promoter AAVs Besides restricting AAV transgene expression to particular...