...materials to the research community. References 1. Coroadinha, A. S., et al. "Effect of osmotic pressure... AAV2 interacts with the packaging cell's cell membrane When packaging adeno-associated viral vectors ...to be due to the viral particle’s affinity for heparan sulfate proteoglycans on the surface of the packaging...supernatant, the viral particles associate with the cell membrane and are taken into the cell via endocytosis. In...looking to improve their AAV yields. Given the wide range of downstream applications scientists rely on our...preparation. Through sugar detection assays, in vitro transduction, and silver stain approaches we show that there...test the endocytosis of a fluorescent dye and transduction of GFP-expressing AAV particles we did not observe...

...the life scientist or bioengineer.   Figure 1: Map of a plasmid with its elements described below... origin of replication (which controls the host range and copy number of the plasmid) and typically include...for further study. Promoter Region Drives transcription of the target gene. Vital component for expression...cell types. Primer Binding Site A short single-stranded DNA sequence used as an initiation point for PCR...vector containing the newly inserted gene is transformed into bacterial cells and selectively grown on...biologists to broadly utilize plasmids across a wide range of applications. Some common plasmid types include...

...plasmids. The Zhang lab, on the other hand, uses a 1 plasmid, single infection system. Genome wide mouse...target cells that have previously been stably transfected for Cas9 expression. The Yusa lab has used their...

...literature? Very often the data of interest are in 1 or 2 figures from one paper and then another figure...preprints, data, software, micropublications) in grant proposals. ReFigures are one such research product...

...could be kept frozen after collection. Figure 1: Paul included a voltmeter on top of the battery box...ahead of time included contacting YNP staff to arrange permits to visit places not accessible to the public...night was fun but also very tiring. It feels a bit strange to be using a micropipette and gloves at a campsite...similar to a type II-C Cas9 from Parvibaculum lavamentivorans, whose optimal growth temperature is 30°C. ...rRNA in bacterial RNA-Seq. This enriches for transcripts of interest without an increase in the number...

...into three main effector functions.  Figure 1: Fc effector functions in an immune response to a ... the pores being opened in the pathogen’s cell membrane (Dunkelberger & Song, 2010). As cells cannot survive... survive when holes are punched in their membranes, the pore'd pathogens quickly lyse and die.  Antibody...where the eosinophils release cytotoxins via degranulation.  Antibody-dependent cellular phagocytosis (...

...do researchers track fluorescently labeled cells? 1. Epifluorescence microscopy This technique uses a ...of the immune system and its dynamics. References 1. Peters, Nathan C., et al. "In vivo imaging reveals...for a growing organism to make the complicated transition from a clump of cells to a fully differentiated...cooperate to cause a higher-energy electronic transition in a fluorescent molecule (see figure to the ...past 15 years efforts have been made to create transgenic mice expressing fluorescent protein reporter ...microscopy enabled scientists to see how chaotic and random the scanning was and how fast T cells could jump...Blue-green fluorescent proteins like EGFP Yellow-orange fluorescent proteins like TagRFP, tdTomato, DsRed...

...targeting MNase to antibody bound proteins (Figure 1). Importantly, since Protein A has weaker affinity...sonification. Though it helps capture weaker or more transient DNA-protein interactions, the reliance on fixation...McKay lab we study how differential access to transcriptional regulatory elements is controlled during development...

...in online patient/caregiver communities  Table 1: Examples of defining your audience for science communication...  For example, while “PCR test” has recently transitioned from jargon to non-jargon in the context of ...number of characteristics. Common ones include age range, familiarity with the subject, shared interests,...

...year and happy reading! Top 10 blog posts of 2015 1. CRISPR 101: Homology Directed Repair Addgenie Chari...used to generate inversions, deletions, and translocations. It also provides researchers with many different...Where Do I Find One? If you need help making a transition into a new job or maybe to an entirely new career...

...Rechsteiner M. Cell. 1982 May;29(1):33-41. PubMed. Protein transduction: unrestricted delivery into all...protein Tat. Although CPP fusions can translocate a cell membrane, the presence of the CPP may alter protein... (78-79% after two rounds of transduction.) Compared to CCP transduction, iTOP is at least four times ..., and cell death post-transduction is low. Neuronal stem cells transduced with iTOP resulting in...lab suggests that primary cells may be better transduced using only protein. Read on for a description...description of the lab’s iTOP protein-only transduction method and its potential applications to CRISPR/Cas9 genome...genome editing.   Protein-only transduction? Niels Geijsen’s group in Utrecht wanted to pursue a nonviral...

...double-stranding step and reduces both the time and cost for AAV genome sequencing.  Figure 1: The...previously require a preliminary double stranding step. The double stranding step was assumed to be necessary...genome is single-stranded and both TA-based ligation and tagmentation require a double-stranded template.  ...extraction, the [+] and [-] strands might naturally associate forming a double-stranded species that could be...extract is a heterogeneous mix of single-stranded and double-stranded species. While not all of the molecules...extract associate with complementary strands, enough double-stranded species exist in the mix to serve as...viral genome We hypothesized that this double stranding step might not actually be necessary. Since the...

... Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds. Cell, 160(1), 339-350 (2015). PubMed...recent paper, "Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds." Adam has...visualize the concepts discussed in the paper. The transcriptional control of multiple loci is deftly coordinated...cerevisiae with few techniques that are easily transferrable across host species or metabolic pathways. A...CRISPR-based RNA scaffolds to mimic natural transcriptional programs on multiple genes. Modifying the ...synthetic RNA scaffolds that can recruit various transcriptional regulators (Zalatan et al. 2015).  These scaffolds...nucleotide sequence then subsequently recruit a transcriptional regulator to modulate expression at that locus...

...heat-fixed smear with the crystal violet stain for 1 minute. Rinse the slide briefly with tap water. Flood...diderms, have a thinner peptidoglycan membrane and an outer lipid membrane. Common gram negative bacteria include...within their membrane, while the stain is washed from the gram negative bacteria’s thinner membrane. Procedure...destroys the outer lipid membrane, and the thin inner peptidoglycan membrane is unable to retain the stain...expanded on Gram’s discovery, adding a second stain, safranin, as a counterstain to help visualize the bleached...or monoderms, have a single thick peptidoglycan membrane. Common gram positive bacteria include staphylococci...and immediately rinse. Flood the smear with the safranin counterstain for 30 - 60 seconds. Rinse the slide...

... Figure 1: Identification and enrichment use of XMAS-TREE. a) Cells are transfected with three different...ready-to-use viral preps   Improved cell-free transcription/translation system to examine biosynthetic pathways... Gabrielle Clouse Recently, cell-free transcription/translation systems (also referred to as TX-TL systems...modifications to edit DNA without requiring a double-stranded break. The Xiao Wang Lab has recently deposited.... These plasmids use “Cas9-mediated adenosine transient reporter for editing enrichment”, or XMAS-TREE...identification of essential MYC binding sites in a wide range of human cells.  A new pooled library from Junjie...

...Tetbow (Tetracycline trans-activator Brainbow) (Sakaguchi et al., 2018) (Figure 1). Once introduced, fluorescent...fluorescent protein (XFP) genes in a transgene. When multiple copies of the transgene cassette are introduced, stochasticity...fluorescent proteins is only possible at low copy number ranges, resulting in low fluorescent protein level. The...gene delivery is more common than conventional transgenic strategies. In such vector-based gene delivery...in the original method, we used the Tetracycline trans-activator system to boost the expression levels,...expression level of XFPs, most likely by suppressing transcription. It is critical to express a minimal amount ...Bio-Protocol 8 (2018): e3046. Livet, Jean, et al. "Transgenic strategies for combinatorial expression of fluorescent...

...construction of redCALWY-1, a red-shifted FRET sensor for zinc using variants of mOrange2 and mCherry as donor...field is a series of fluorescence resonance energy transfer (FRET)-based calcium biosensors. Perhaps most ...developed and published in 2010 for visualizing the transcription factor cAMP-responsive element-binding protein...really important and when? That is not known.” Transition Metals Maarten Merkx of Technische Universiteit...particularly for the intracellular imaging of transition metal ions, such as zinc, copper, and iron. “...the sensors involves plenty of trial and error. Transition metals also present challenges in that they are... way to make a FRET sensor with a large dynamic range,” he says. Merkx recently applied this “trick” to...

...preferentially). You can also rank the plasmids by abundance, scaled to 1, to create a “cumulative wealth... seems straightforward: E. coli bacteria are transformed with the library DNA, they grow and replicate... Each Addgene library has its own depositor transformation and amplification protocol, but the overall...Maintain representation of the library during transformation First, use enough DNA and competent bacteria...and we use electroporation to ensure a good transformation efficiency. For a typical library, Addgene ...growing the cells on solid plates, where each transformant forms its own colony, uninfluenced by the growth...concern, but Addgene still recommends plating the transformation mixture on solid media. The colonies are then...

.... You can email her at [email protected].   References 1. Huang, Ally, et al. "BioBits™ Explorer: A modular...that green fluorescent protein through transcription and translation. Unfortunately, having live cells in... like gel electrophoresis to separate DNA or transforming cells with green fluorescent protein, these ... into a host cell (through a process called transformation), essentially giving the cell all the instructions...enzymes that can convert certain chemicals into fragrances that they can smell, or enzymes that can create...perhaps consider using some elements of BioBits to transition your undergraduate students from the classroom...

... contain the full length plasmid.   Figure 1: The full lentiviral plasmid is expected to be >12...recombination, one resulting plasmid carries the transgene of interest (aka the viral genome) and the other...resistance gene and origin will replicate, and the transgene will be lost. Over time, bacteria containing the... grow! Our 5 Tips for Troubleshooting Viral Transductions will help with the next steps.   References...recombination of plasmid DNA without affecting mRNA translation efficiency or half-life. RNA 25:507–518. https...common lab E. coli strains Find tips for viral transduction  ...