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...breakdown Intro Chapter 1 - How Joachim ended up at the University of Amsterdam Chapter 2 - Joachim’s Academic... in microscopy. Chapter 7 - Open access in biology and how it speeds science. Chapter 8 - The importance...your passion and pay attention to the details. Chapter 3 - Answering interesting biological questions ...Joachim’s lab. New microscopy probes and more. Chapter 5 - mTurquoise II and the creation of improved ...improved fluorescent proteins through mutagenesis. Chapter 6 - The future of FRET and fluorescent protein tools...microscopy (University of Amsterdam). He develops, characterizes and uses genetically encoded fluorescent probes...

... Empower and Accelerate Research Discoveries) Challenge provides grants for rare disease research. This...result in global creatine deficiency, which is characterized by development delays, speech delays, seizures...video. “SLC6A8 belongs to a large family of solute channels that are responsible for maintaining the nutrient... Scientific Advisor. It’s possible that solute channels typically expressed on other cell types can compensate...for or bypass the dysfunction of another solute channel. With the BeHeard award, the ACD will use Addgene...screen in mammalian cells to identify any solute channels that can compensate for the defect caused by SLC6A8...collection consists of human and murine solute channels, the team will be able to validate their findings...

...while writing each section. Research challenge The Research Challenge motivates the request for application...the research challenge. Demonstrating expertise in the field and past successes boost chances in receiving...how you, the expert, will address the research challenge. Now is the time to make bold statements. While...how your innovation will address a variety of challenges and impact the field - its encouraged to dream... funded. Budget The Budget gives the writer a chance to delegate funds to supplies, personnel, equipment...time budget writer, you may find this section challenging because it’s hard to determine the exact value... differs from publication writing and may be challenging to master at first. Additionally, reach out to...

...have sought to alleviate many of these challenges by exchanging the fluorescent donor for a bioluminescent...tissue-penetrating wavelength above 600 nm. LumiFluors Schaub, F.X., et al. (3) designed two imaging probes. ...When the responsive protein binds to calcium it changes conformation and brings the two fluorescent proteins...concentrations of biological molecules. FRET sensors face challenges of photobleaching, autofluorescence, and, in ...cyan-excitable donors, phototoxicity. Another challenge to using FRET sensors comes when employing optogenetic...create a calcium sensor with the following characteristics: No photoexcitation High Ion specificity High...and NanoLuc® Luciferase. CalFlux VNT responds to changes in intracellular calcium and is useful for direct...

...biological devices. Chapter 6: - Making research go faster through Open Access. Chapter 7/Outro: Tom’s Involvement...grad school to a startup and back to academia. Chapter 1: How working at a startup prepared Tom for an...work-life balance in Academia. Will it all pay off? Chapter 2: The Centre for Synthetic Biology and Innovation...training, startups, and real applications of SynBio. Chapter 3: Synthetic biology as the engineering of biology...standardized, disciplined, and open techniques. Chapter 4: Research projects underway in the Ellis lab ...microbial cooperation, and antibiotic production. Chapter 5: The future of Synthetic Biology. Wide adoption...Working at a SynBio Startup in Our Interview with Michael Koeris Resources at Addgene.org Find Plasmids...

...simultaneously characterize the ‘social networks’ of 157 super enhancers.  3. Measuring temporal changes in chromatin...that surround changes in gene expression and determine who’s a key driver of these changes. By generating... CAPTURE method, the Xu Lab made the following changes to create CAPTURE 2.0:  Replace the biotinalatable...chromatin interactions at a promoter in the well-characterized beta globin locus. The two methods had similar...Chromatin interactions at five sites in the well-characterized beta-globin locus were analyzed with CAPTURE...application for CAPTURE 2.0 is detection of temporal changes in chromatin conformation, like those that occur...When chromatin conformation data is compared with changes in gene expression, chromatin accessibility, and...

...through my university’s purchasing system. What’s the order status? My purchasing department faxed the PO...website? Many vendors will process orders based on a purchase order, which is an official document listing materials...email, or orders that are placed through internal purchasing systems. All orders must be placed directly on...create an Addgene account and place an order: a purchasing agent, lab manager, or the end user. All updates... checkout, you will have the option to “Pay by Purchase Order.” Enter the PO number you generated through...directly if we require a copy of your PO. If your purchasing department requires a quote in order to generate...check out our Help Center article How do I pay by purchase order (PO) number? You can find answers to other...

...mediate a C to T change (or a G to A change on the opposite strand). ABEs make an A to G change (or a T to ...out of 12 possible base-to-base changes. The other 8 base-to-base changes require Base Transversion Editors...to C change on the opposite strand). Both are base transition editors: converting one purine base to the...purine-to-purine or pyrimidine-to-pyrimidine base changes. Solid red arrows indicate a conversion directly...the base editor. Dotted red arrows indicate the change introduced to the opposite strand following base...dCas9 prevents base excision repair, which would change the U back to a C base. To increase base editing...convert adenosine to inosine, resulting in an A to G change (Gaudelli et al., 2017). Creating an adenine base...

...expression of receptors, ion channels, and growth factors resulting in changes to the cell line’s growth ...medium. Daily medium exchanges help alleviate cellular stresses associated with pH changes and resource consumption...In addition to benefitting your cells, medium exchanges may also impact the quality of the lentiviral ...harvest. Studies have demonstrated that extreme changes in osmotic pressure and pH can destabilize the ... the electrostatic repulsion of the negatively charged cell with the virus. To circumvent this problem... transductions typically employ the positively charged polycation, polybrene. Interestingly, one study...conditions for specific applications, stick with them! Changes in such variables as cell density, volume, incubation...

...number. Here, we will consider the replication mechanism of plasmids under stringent control, such as pSC101...expression, so high levels of DnaA act as a feedback mechanism to reduce further transcription. Replication is...for ATP to re-activate DnaA. These regulatory mechanisms allow E. coli to control chromosomal replication..., it can be regulated indirectly via the same mechanisms that affect DnaA and IHF. RepA also controls ...needs to get before handcuffing occurs. These mechanisms to regulate plasmid replication through the dual...Figure 3: Comparison of replication regulation mechanisms of stringently controlled plasmids pSC101 and... or sequences necessary for initiation or the mechanisms that regulate their replication, but their copy...

... of light-gated potassium channelrhodopsins, named Kalium channelrhodopsins (KCRs), from a fungus-like...Govorunova EG, et al. Kalium channelrhodopsins are natural light-gated potassium channels that mediate optogenetic...-2P Novel class of light-gated potassium channelrhodopsins Myc-tag antibody anti-c-Myc [9E10] now available.... The resultant JEDI-2P sensors overcome many challenges in 2-photon in-vivo imaging of voltage fluctuations...   Novel class of light-gated potassium channelrhodopsins by: Mike Lacy New work from the labs of Mingshan... attempts to engineer potassium-selective channelrhodopsins. HcKCR1 has a maximum absorption of 540 nm...

...cycle of one step. Challenges in the LEGO science video process One major challenge when we started was...Gnome-Proof" This is why the “Bricking Science” YouTube channel was born. The name came from LEGO bricks + science...will bring people together and make these daily challenges a little easier to overcome. We want to encourage...motion format for our videos as it is the most approachable for LEGOs. Stop motion consists of taking many... no pre-made LEGO laboratory sets so we had to purchase the big LEGO classic box, the Big Bang Theory ...gotten easier now that I identify with the main character in the video. We’ve grown attached to the work...“WOW, now I have my own laboratory” (the main character is much more than a piece of plastic for us now...

...to a negatively charged residue, aspartate or glutamate, imparting the negative charge associated with...carry only a single negative charge as opposed to a double negative charge in the phosphate group. Additionally... acids fail to recapitulate the true steric and charge-based nature of the phosphoryl-modification; they...phosphorylated intermediate of a protein for characterization. However, phosphoproteins are often low abundance...incorporate phosphoserine A Sep-tRNA synthetase, which charges this tRNA with phosphoserine A specialized elongation...an optimized elongation factor that accepts Sep-charged tRNAs during translation. Many ‘flavors’ of Sep-OTS...et al. "RNA-dependent cysteine biosynthesis in archaea." Science 307.5717 (2005): 1969-1972. PubMed PMID...

...generate the strictly monomeric EGFP variant, mEGFP (Zacharias et al., 2002). GFP needs oxygen Chromophore formation...incorporation into proteins are steps in that direction (Chatterjee et al., 2013; Hilaire et al., 2017). GFP can ...specific RNA stem loop-structure (Querido and Chartrand, 2008). Finally, a nuclease-deficient Cas9 can...post-translational modification with fatty acyl chains. For instance, myristoylation requires an N-terminal...microscopy (University of Amsterdam). He develops, characterizes, and uses genetically encoded fluorescent probes...on twitter: @joachimgoedhart.   References 1. Zacharias, David A., et al. "Partitioning of lipid-modified...of Green Fluorescent Protein Fused with a Single Chain Fv Protein." Journal of Biological Chemistry (2000...

... Hirsch ML, Li C, Bellon I, Yin C, Chavala S, Pryadkina M, Richard I, Samulski RJ (2013) Oversized AAV...transgene to reconstitute the full-length transgene (Chamberlain et al., 2016). It’s generally accepted that some...occurs via this region of overlap, but the exact mechanism is not yet known. Drawbacks:  Generally an ... either the overlapping or the trans-splicing mechanism. The splice site donor and acceptor sequences ... . https://doi.org/10.3389/fnins.2017.00503  Chamberlain K, Riyad JM, Weber T (2016) Expressing Transgenes....2015.140 Frischmeyer PA (2002) An mRNA Surveillance Mechanism That Eliminates Transcripts Lacking Termination...Lai Y, Yue Y, Liu M, Ghosh A, Engelhardt JF, Chamberlain JS, Duan D (2005) Efficient in vivo gene expression...

...PMC4923196. Panchapakesan, Shanker Shyam S., et al. "Ribonucleoprotein purification and characterization using...RNA is not inherently fluorescent and can be challenging to track intracellularly, but aptamers permit...that bind metabolites, leading to conformational changes that control the expression of coding RNA. Spinach-based... bind DFHO and fluoresce in the red and orange channels, respectively. Both Red Broccoli and Orange Broccoli...orange (TO) and fluoresces in the orange or red channel, depending on the structure of the TO derivative... used Mango to study RNA-protein complexes (Panchapakesan et al., 2017) and short non-coding RNAs (Autour...Hauer, and Diane E. Griffin. "Development and characterization of sindbis virus with encoded fluorescent ...

...construct expressing channelrhodopsin 2 (ChR2), a blue light sensitive cation channel, fused to mCherry ...emitting diodes, thermal sensors, and microfluidic channels. These features enable the fibers to emit light...stimulation of its associated fibers across multiple channels and can also record tissue temperature proximal...considerations of actually using it - the device has a rechargeable battery, is relatively small, and can be detached...Sands, J., Sahasrabudhe, A., Anikeeva, P., & Chandrakasan, A. P. (2020). Modular Optoelectronic System...Garwood, I. C., Yan, W., Niroui, F., Fink, Y., Chandrakasan, A., … Anikeeva, P. (2023). Multifunctional ...Addgene blog Plasmid tools for microbiome studies Channel rhodopsins for optogenetics Viral Vectors 101: ...

...isotype? Antibodies are composed of light chains and heavy chains made up of variable regions and constant...regions, just the heavy chain constant regions or solely the Fc region may be changed.   Chimeric antibodies...antibodies can help A chimeric antibody is made by changing an antibody’s natural isotype to that of a different...will react with the same secondary. If you were to change one of the antibody’s isotypes, however, you could...be converted? Isotype conversion is a unique characteristic of recombinant antibodies. Recombinant antibodies...containing all constant regions, just the heavy chain constant regions or solely the Fc region of a different...

...sufficient to prevent any pH change, as we still observed a slight change in pH (from 7.4 to 6.8). It’...tubes (Fig. 1). It’s important to note that the change in pH only occurred when the solution thawed, as...samples were frozen. This was clear by the gradual change in pH visible at the sample interface upon thaw...found that 30 seconds of venting attenuated the pH change caused by storage on dry ice (Fig. 3). However,...CO2 to leave the tubes, and would prevent the pH change upon thaw (Fig.4). To test this, we froze the tubes...stability (Murphy et al., 2013). However, the pH changes in our solutions are transient and return to normal... have no evidence to suggest that the transient change in pH affects AAV function. Furthermore, another...

...cells. These changes were larger than previously reported for other organisms, and the Gottschalk group hypothesizes...screens, which enables researchers to maximize the chance of transducing a only a single gRNA per cell. The... Genetically encoded voltage indicators (GEVIs) change fluorescence in real time when neurons or muscle...technical difficulties such as small fluorescence changes or a requirement for very high excitation light...these indicators difficult to work with.  The Gottschalk lab has surveyed a range of microbial rhodopsin... that can be used to monitor real time voltage changes in worms and have made these constructs available...detect action potentials in worms with fluorescence changes of up to 128% per 100 mV in the pharynx, and 78%...

...Chu H, Chan JF-W, Yuen TT-T, Shuai H, Yuan S, Wang Y, Hu B, Yip CC-Y, Tsang JO-L, Huang X, Chai Y, Yang...H-W, Cai J-P, Chan W-M, Ip JD, Chu AW-H, Zhou J, Lung DC, Kok K-H, To KK-W, Tsang OT-Y, Chan K-H, Yuen K-Y...in a publication by Xiuyuan Ou, et al. that characterized the spike (S) protein of SARS-CoV-2 (Ou et al...Jackson Laboratory. Originally developed and characterized by Stanley Perlman, of the Carver College of...for the advancement of scientific research, and mechanisms to maintain and grow these critical organizations...Chen J, Hu K, Jin Q, Wang J, Qian Z (2020) Characterization of spike glycoprotein of SARS-CoV-2 on virus...

...fit for me, then I didn’t need to explain why I changed jobs. I highly recommend taking on a similar position...become bored because I was not being mentally challenged. For these and other reasons, I started  looking...this company for several years, I was ready for a change.  Working abroad: Research in Australia I then ...for a 6 month project to validate some genomic changes, so this arrangement was ideal for everyone.  The...combination of genetic mutations. This is a particular challenge in pancreatic cancer where one drug does not work... fit in with the company and that my ideas and changes were welcomed, which is a benefit over larger companies...who are grateful for some help.  I also get the chance to contribute content to the Addgene’s Website ...

...goals can be a real challenge. On top of that, in the midst of a rapidly changing, globalized environment...training in a globalized world where individuals can exchange ideas, network, collaborate, and innovate internationally...technical and logistic support to increase the chances of success, but also nurture a supportive and protective...mentees with a broader, first-hand vision of the challenges and career opportunities that truly exist outside...international mentor creates a closer and more approachable relationship for the mentee, where (s)he feels...was much more than that. I discovered that the challenges that I am facing in my career are common problems... that many others have faced yet, still, this challenge did not hinder them from advancing their careers...

...deaminase. Base changes from C•G-to-T•A are mediated by cytosine base editors (CBEs) and base changes from A•T-to-G...capabilities. Base editors make specific DNA base changes and consist of a catalytically impaired Cas protein...target site, and the deaminase induces the DNA base change without a DNA double-strand break. But base editors...is that the editing domain must induce the base change before the Cas domain lets go of the DNA. ABEs ...hold on” long enough to the DNA to induce a base change. Because of this, ABEs are only weakly compatible... of the fetal hemoglobin gene, ABE8e made both changes simultaneously with 54% efficiency, about 7 times... DNA base swaps, so called point mutations that change C to T, G to A, A to G, and T to C, represent about...

...set-up has been changed (for instance after replacing a broken lightbulb or exchanging excitation filters...fluorescent proteins may display reversible intensity changes (Shaner et al, 2008; Bindels et al, 2017) and photoswitching...fluorescence signal, showing no or little deterioration or change of the signal during the course of the experiment...and by comparing how the fluorescence intensity changes over time, the photostability of the fluorescent...comparable (lower panel of figure 1). To avoid changes in excitation power, it is important to perform...filters). This avoids substantial changes in excitation power and will help to keep the photostability of...microscopy (University of Amsterdam). He develops, characterizes and uses genetically encoded fluorescent probes...

...Other PCR conditions can be changed to further lower fidelity, like changing the metal cofactor and the...evolution. This is a larger process of gradually changing and selecting the best antibodies for a specific...crystallizable; IgG: immunoglobin G; ScFv: single-chain fragment variable; sdAb: single-domain antibody ...Directed evolution is a method employed to rapidly change a protein towards a specific goal, like improved...style. Instead of leaving sequence generation to chance, you start with a known or predicted structure ...can shrink your mutant library and increase the chances of finding beneficial mutations. In other words...antibodies developed by IPI are extensively characterized and validated, with the application data from...

...McClinntock who first discovered transposons due to changes in coloration of corn kernels. Aside from corn,... or a reverse transcriptase, depending on the mechanism of transposition.  Target site: Different transposons...elements are split into two classes based on their mechanism of transposition: Class I TEs, also called retrotransposons...retrotransposons. They transpose by a ‘copy-paste’ mechanism (Fig. 1). They first copy themselves as RNA transcripts...like retroviruses both in their structure and mechanism of replication. They contain two genes: gag and...Retrotransposons are mobilized by a ‘copy-paste’ mechanism. First they copy themselves as RNA transcripts...transposons have a non-replicative “cut and paste” mechanism of transposition: they first excise themselves...

... ́-phosphate to the ligation). The structural/mechanistic reason for this differential is not known for... E., et al. "DNA ligases: structure, reaction mechanism, and function." Chemical reviews 106.2 (2006):...PMID: 2753355. 12. Barany, Francis. "The ligase chain reaction in a PCR world." PCR Methods Appl 1.1 (...PMID: 1842922. 13. Wiedmann, M., et al. "Ligase chain reaction (LCR)-overview and applications." PCR Methods... PubMed Central PMCID: PMC1933048. 16. Cheng, Changming, et al. "Electrochemiluminescent assay for detection...detection reaction/CGE method using a LIF dual‐channel detection system for direct identification of allelic...reaction-fluorescent microsphere assay for characterization of resistance-mediating polymorphisms in African...

...engineering. Chapter 1: Early research on CRISPR and what it's like to work in a competitive field. Chapter 2: ...Institute of Singapore. Chapter 3: Biology research and resources in Singapore. Chapter 4: Life as a new primary...project ideas, applying for grants, and mentoring. Chapter 5: Wei Leong Chew's lab management style and encouraging...encouraging mentees to pursue cool projects. Chapter 6: Projects in Wei Leong Chew's lab. Genome engineering...engineering, gene delivery methods, AAVs, and more. Chapter 7/Outro: Advice for researchers pursuing careers...

... the common cytokine receptor gamma chain (c) and is characterized by a lack of immune cell development... cargo (DNA or RNA) to target cells has been challenging. A variety of vector delivery technologies have...repair via homology directed repair (HDR). This characteristic makes AAV vectors especially amenable to correcting...caused by mutations in the gene RPE65. It is characterized by childhood onset of blindness and was thought...treatment appears to be both safe and effective. Challenges for AAV gene therapy Although AAV-based gene ...promising, scientists are working to overcome key challenges to their efficacy:  Pre-existing immunity: Many...10.11 (2003): 964-976. PubMed PMID: 12756417. 5. Chan, Ken Y., et al. "Engineered AAVs for efficient noninvasive...