...and open a new conversation about GMOs.  Challenge #2: Technical obstacles to plant synthetic biology In...transformation to testing and a lack of well-characterized genetic tools make it challenging to engineer...understood. Additional work has also identified and characterized mechanisms and components of the circadian rhythm...

...IgM, IgD, and IgG are deleted.     Figure 2: Isotype switching requires DNA recombination of the...isotype? An isotype determines several key characteristics of an antibody as well as the role it plays... regions result in several unique physical characteristics of these five isotypes, such as the number ...IgG4. IgE IgE antibodies help protect against parasites but also play a role in allergies. They are found...

...divided by the off-target activity. Figure 2: Specificity is the measure of off-target activity...experimental approaches. One example is CasX, characterized by the Oakes and Doudna Labs, which is both ...research and potentially treatments. Cas12b, characterized by the Zhang (Strecker et al., 2019) and Li ... relative to Cas9. As scientists continue to characterize CRISPR nucleases from a variety of sources, ...

...JW, Cammarata M, Perez M, Agarwal P, Brodbelt JS, Legube G, Miller KM Genes Dev. 2015 Jan 15;29(2):197...essential for the structure of paraspeckles. Clemson CM, Hutchinson JN, Sara SA, Ensminger AW, Fox AH, Chess...

...3570-5. doi: 10.1073/pnas.1420294112. Epub 2015 Mar 2. PubMed. Find the plasmids from this publication ...from Pseudomonas aeruginosa to cleave 4 gRNAs separated by 28 base Csy4 recognition sites from a polycistronic...protoplasts. Each pair of gRNAs targeted genomic sites separated by 350-750 bp, allowing them to examine the frequency...different biological systems. Although Xie et. al characterized the system in plants, PTGs will likely work ...

...is the most charged, with 5 negatively charged and 2 positively charged amino acids. FLAG tag has been ...the need for an antibody. Tags can enable the separation of detection of a mutant protein vs. endogenous...

...automated. Unlike gel electrophoresis, you only need 1-2 ul of sample and the run time is just a few minutes...micro- or nanofluidic channel and the sample is separated by electrophoresis. Smaller fragments migrate ...

...two options to begin your plasmid deposit. Figure 2: Choose to submit plasmid(s) online or by using a ...format. Figure 6: Add sequence, maps, and files. Step 2: Gene and Insert *Note: This page will not appear ... into it. You will have the option to indicate separate promoters, tags, or cloning information for each...

...156–159. https://doi.org/10.1016/0003-2697(87)90021-2 Chomczynski P, Sacchi N (2006) The single-step method...gradients and ultracentrifuged for up to 20 hours to separate the intact RNA from DNA and proteins. Although...and Sacchi showed that RNA could be effectively separated from DNA and proteins by a simple extraction protocol...thiocyanate solution. However, instead of RNA separation using cesium-chloride gradients, water-saturated...ultracentrifugation!), the layers of phenol and chloroform separate, and RNA is retained in the top, aqueous layer...

...still depolarize neurons at lower MOIs. Figure 2: ChRgers are delivered by intravenous injection into... “designer” channelrhodopsins with desired characteristics. The model was trained to predict if a channelrhodopsin...these remaining sequences were selected for characterization with patch clamp electrophysiology. All thirty...named ChRger1, ChRger2, and ChRger3, were then characterized further by expression in cultured neurons and...performed well in a tissue culture dish, that doesn’t guarantee their success in vivo. Therefore, the Arnold and...

...main decisions to make: 1. Which modification? and 2. Which viral vector should be used? Modifying gene...need to be given some time to adapt to a testing apparatus. General stress levels of the test animal may ...the humans’ audible spectrum. The experimental apparatus should be cleaned thoroughly between animals as...beginning. Variability source Standardization parameters Animal Strain, age, sex, housing conditions...order to maximize possible effects. Injection Parameters: The injection needle should be as thin as possible...

..., but at a lower efficiency (~20%). Figure 2: Schematic of multicomponent assembly with SLIC. Multiple... other dNTPs. Once the vector and insert are (separately) digested to create 10–12 base “chewed back” ... T4 polymerase (left branch); by annealing two separate PCR products (middle branch); or by incomplete...

... and transfer 100 µl of glycerol stock to each of 2 96-well microplates (identical copies, see image above...for processing several sequencing reactions in parallel. We have a few tricks to simplify the process....saves us the step of purifying the DNA and gives comparable success rates and sequence quality, which is ...

...retroviral library was used to transduce T helper type 2 (Th2) cells from mice that constitutively express ...to manipulate genetically. To solve this issue, Sarah Teichmann and colleagues created a retroviral CRISPR...

...work with Grows extremely fast (doubling time of ~ 2 hours) Amenable to genetic manipulation The first...10.3389/fmicb.2020.607028  Riley, R., et al., Comparative genomics of biotechnologically important yeasts...

...Without binding to the guide RNA, Cas9 cannot cut. 2. Bind to target DNA in the presence of a guide RNA...bacterial genomes consist of repeated elements separated by unique sequences. When researchers first discovered...

...to visualize multiprotein complexes.  Fig. 2. Multiplex immunolabeling with subclass-switched recombinant...and validation pipeline. (B) Schematic shows the separate elements of the R-mAb expression plasmid involved...

...and in line with Addgene style.        Fig. 2: Reference image (left) provided to our graphic designer...the antibody guide always gets an expert review, separate from the author, and a second review from someone...

... adapted to a competitive format. Figure 2: Overview of using Competitive ELISA to quantitatively... wells with a purified protein allows you to characterize properties of an antibody that recognizes that.... As mentioned above, it can also be used to characterize antibodies against the antigen bound to the ...

...protocols 4.4 (2009): 495-505. PubMed PMID: 19300443. 2. Geraerts, Martine, et al. "Comparison of lentiviral...if preparing several different lentiviruses in parallel, this approach should be considered. Similar to...consider using a reverse transcriptase mutant in a separate control reaction. This will allow you to quantify...

...products have distinct localizations. Figure 2: Split-wrmScarlet labeling proteins with different... than split-sfCherry3. It was created by Maria Ingaramo, Jerome Goudeau, Cynthia Kenyon, and colleagues...Cas9-Cas12a gRNAs under a single U6 promoter. The paralog and dual-targeting hybrid gRNA library can be used... used to identify genetic interactions between paralogs. The exon-deletion hybrid gRNA library deletes...

... Cure for HIV/AIDS. Int J Mol Sci. 2023 Jan 13;24(2):1563. doi: 10.3390/ijms24021563 Resources on Addgene.org... therapeutic potential in mice with a dominant cataract disorder caused by a single copy of a gene known...known as Crygc (Wu et al). They found that the cataracts phenotype could be rescued by zygote co-injection...polymorphisms (SNPs) in the TTR gene and is molecularly characterized by misfolding of the TTR protein, which contributes...

...more tips on using social media as a scientist here. 2) Public engagement - Participate in science festivals...book.  Many thanks to our guest blogger, Sarah Schmidt! Sarah Schmidt is engineering resistance against...This post was contributed by guest blogger Sarah Schmidt, a Marie Curie Fellow at The Sainsbury Laboratory...

... to remove this default setting. Timesaving Tip #2: blastx searches are inherently slower than blastn... BLAST button, consider the Program Selection parameter, as this will affect the amount of time to perform...BLAST button, you may have noticed the “Algorithm parameters” link. Click on this link to view advanced BLAST...have to remember to adjust any blastp algorithm parameters. We use the default scoring matrix BLOSUM62, ...

... those that also infect the liver.    Fig. 2: Summary of the generational evolution of the AAV ...SSAP system reports an efficiency of up to 20%, comparable to commonly used CRISPR/Cas9 methods for large...

...Era of Personalized Medicine. Genes Dis. 2017 Jun;4(2):43-63. DOI: 10.1016/j.gendis.2017.04.001 Maetzig.... 2011 Jun;3(6):677-713. DOI: 10.3390/v3060677 Varanda C, Félix MDR, Campos MD, Materatski P. An Overview...

...PubMed PMID: 8491198. PubMed Central PMCID: PMC413431. 2. Bukrinsky MI, et al. “Active nuclear import of human...We will be covering these safety measures in a separate, upcoming post. If a viral vector seems like ...

... high-priced commercial devices.     Fig. 2: Workflow for open software (left) vs. open hardware...in developing better, more sustainable, easily sharable products. Open-Source Microscopy Sharing Hardware...

...of human codon-optimized  cryptochrome 2 (CRY2) from Arabidopsis thaliana. When exposed to blue light, ...protein. After fusing the CRY2 and CIB1 domains to separate proteins of interest, a researcher can cause them...

...dimerization-dependent protein interactions. FEBS letters, 591 2, 415-424. PubMed PMID: 28032891. This is a different...feature is particularly useful for in-depth characterization of dynamic protein complexes. Split-BioID,...of the bait proteins’ interactome. Further characterization of these candidates is critical to understanding...

... already have an end product in mind, steps 1 and 2 will generally be straightforward. Likewise for step...present in a single lentiviral transfer vector or separate transfer vectors. May contain reporter gene (e.g...model.  If your expression system is not well characterized in terms of CRISPR use, you will want to invest...

...GUS-staining is a very useful visual reporter (Figure 2). The GUS enzyme cleaves a substrate, which will then...vincristine, are regulated in the medicinal plant, the Catharanthus roseus (Figure 1). The ultimate goal of our ...   C. roseus is not a model organism such as Arabidopsis or tobacco. Studying gene function in C. roseus...method for overexpressing genes In plants like Arabidopsis and tobacco, Agrobacterium infiltration into ...expression assay by agroinfiltration of leaves." Arabidopsis Protocols. Humana Press, 2006. 225-229. PubMed...transient gene expression analysis in seedlings of Arabidopsis and other plant species." Plant methods 5.1 (...expression method for analyzing gene function in Catharanthus roseus seedlings." Frontiers in plant science...

...system (by measuring the fluorescence intensity); and 2) to visualize the localization of the FP (fused to...experimental parameters (e.g. excitation light intensity, pH, or temperature). pH Stability: This parameter is ...Photostability can be affected by experimental parameters (e.g. excitation light intensity, pH or temperature...

...the necessary steps to perform:      Fig. 2: Quick-stepping to a FASTA file   With our outline...QC'ing data at Addgene is like standing under a Niagara Falls sized rainbow   Over the years we have...pipelines, including bioinformatics. We loved the parallels between Seqera’s mission and our own, as both ...the sequential tasks in our pipeline, we wrote a separate Nextflow process. In Nextflow, a process defines...concept of channels and implemented the input declaration correctly, the script block in the second process...

...inside the cell,” says Pedre Pérez. Figure 2: Micrographs of the biosensor expressed in the human...colleagues Filip Tomaska and Tim Hanson, are characterizing variants of iGluSnFR3 in vivo and have used...

...You’ve probably heard that only 2% of our genome is made of protein-coding genes, and you might be wondering...transcriptome engineering References Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A Programmable...CRISPR-Based Approaches for the High-Throughput Characterization of Long Non-Coding RNAs. Non-Coding RNA. 2021...

...overview of these new variants:   Figure 2: Inner mitochondrial membranes imaged with structured...and screening that led to identification and characterization of AAV.CAP-Mac. Image reproduced from Chuapoco... Endothelial-specific AAV delivery By Cythina Arokiaraj (Gradinaru lab) Another useful viral tool deposited...

...) 12 (>100) xCas9(3.7)-ABE 69 (1.4) 43 (2) 21 (3) Not examined 16 (>100) Average percent...xCas9 3.6 displayed the second best PAM range. Characterizing xCas9 in mammalian cells Hu et al. compared...SpCas9. Hu et al. performed GUIDE-seq analysis to characterize genome-wide off-targets, since increasing PAM...

... Werner S, Marillonnet S. PLoS One. 2011 Feb 18;6(2):e16765. doi: 10.1371/journal.pone.0016765. PubMed...few new ones that are not used so much but have comparable expression levels to things people do use a lot... developed mostly with model plants in mind, Arabidopsis and Nicotiana benthamiana. N. benthamiana is ...

...strain (which will be covered in more detail in part 2 of this blog series) controls the cell types in which...derived from random crosses between mice of uncharacterized backgrounds to create a limited but genetically...

...company culture.  It’s also a good idea to prepare 1-2 unique questions for each interviewer by looking at...of the theme of my interview stories as the characteristic which the story demonstrates I possess.   Bullet...

...of these issues are not particularly new. Chapter 2, “The Professional Scientist,” addresses the challenges...over two decades, attempts to answer it in five paragraphs, with examples. If that sounds ambitious, that...them wanting to learn more. There are individual paragraphs that may have warranted their own chapters, and...

...mazF gene and a gene for Zeocin resistance (Figure 2). This MazF-ZeoR cassette is flanked by two direct...or diluted as the cell grows resulting in the appearance of unmethylated sites in the chromosome. The ... without plasmid addiction systems. For example, Cara Morin and James Kaper used the hok/sok system to...223.1 (1992): 41-54. PubMed PMID: 1370544. Morin, Cara E., and James B. Kaper. "Use of stabilized luciferase-expressing...

...al (Nature. 1988 Jul 28;334(6180):320-5.) PubMed. 2. A segment of the 5' nontranslated region of encephalomyocarditis...multicistronic vectors simultaneously express two or more separate proteins from the same mRNA. We've discussed promoters...at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next...

...that numbers are not switched around or misread.  2. Track the container - It is just as important to ...to fulfill orders.  The barcoded tubes help to guarantee that no samples get lost during their journey....Addgene Get help with plasmid transformation, preparation, and analysis from our protocols and molecular...

...Hoogsten base pairs and wobble pairs.  Figure 2: (A) The codon wheel depicts the 64 codons that encodes...https://doi.org/10.1038/nsmb1031 Gustafsson C, Govindarajan S, Minshull J (2004) Codon bias and heterologous...3645–3650 https://doi.org/10.1073/pnas.0909910107 Varani G, McClain WH (2000) The G·U wobble base pair. ...

...PMID: 18478103. PubMed Central PMCID: PMC2364656. 2. Dittmar, Kimberly A., et al. "Selective charging ...PMCID: PMC552224. 4. Gustafsson, Claes, Sridhar Govindarajan, and Jeremy Minshull. "Codon bias and heterologous...: 346-353. PubMed PMID: 15245907. 5. Maertens, Barbara, et al. "Gene optimization mechanisms: A multi‐...

...Henrietta Lacks. New York: Crown Publishers, 2010. 2. Gold, Michael. "A conspiracy of cells." State University...line identity and purity making it easier for comparable studies to be performed on a cell line even decades...preferably some of these vials will be stored in a separate backup liquid nitrogen store to prevent loss from...

...non-specific binding in our IHC condition.   Fig. 2: Schematic of the goldilocks antibody selection process...cross-reactivity (binds to non-target proteins), variability (separate batches can perform differently – happens when...

...unc- 119 + selection." MicroPublication 1 (2018): 2–3. Wang, Han, et al. "cGAL, a temperature-robust GAL4...sequencing of neurons from each class to fully characterize the transcriptome. The first data release for...