...initiation proteins to start their own replication. In general, these plasmids tend to be low copy number. Conversely...at the low-affinity sites, the proteins work cooperatively to separate nearby AT-rich regions of DNA. In...are occupied by ATP-bound DnaA, the proteins cooperatively can separate the DNA strands. Created with BioRender.com... from the ori. In the elongation phase, a DNA polymerase synthesizes a new DNA strand at each replication...controlling the initiation of replication. Once the average copy number for a given plasmid has been reached...called iterons located near the ori and directly interacts with DnaA, stabilizing its binding and contributing...negatively regulate its own expression and thereby the overall replication of pSC101 (del Solar et al., 1998)....

...means they can be used with or as fluorophores in several different ways. Researchers have used oligonucleotides...can be created in many different ways, but the general premise is that an aptamer in its unbound state...cells. The new aptamer also has a higher melting temperature than its Spinach ancestors, and has a lower magnesium...fluorophores, including: Environments with higher temperatures, since proteins can denature while aptamers ...likely to trigger immunoresponses. Nucleotides, in general, are far less immunogenic than proteins. Assays...ions or particularly small molecules that do not generate an immune response. Aptamers can be selected for...Peixuan AND Haque, Farzin. RNA Nanotechnology and Therapeutics. Boca Raton: CRC Press, 2014. 5. Hayat, Akhtar...

...your viral vector, you use the same protocol for generating lentivirus, but you use a different envelope ...terminals and can’t be used for neuronal tracing or peripheral gene delivery. Combining the rabies glycoprotein...Cells Are Inactivated by Human Serum. Molecular Therapy 2:218–222 . https://doi.org/10.1006/mthe.2000.0116...Modification of Hematopoietic Cells for Research and Gene Therapy. Viruses 12:1016 . https://doi.org/10.3390/v12091016...Lentiviral Vectors: One Vector, Many Guises. Human Gene Therapy Methods 28:291–301 . https://doi.org/10.1089/hgtb...transport and access to the nervous system after peripheral delivery. Human Molecular Genetics 10:2109–2121...envelope glycoprotein show increased stability in sera and augmented transduction of primary lymphocytes...

...base-pair mCherry insertion. Editing of these motifs generates lesions that are subject to excision and ssDNA...This edit changes the histidine to a tyrosine generating a GFP variant (GFPY66) that has a shifted emission...Reporters in Gene On include a mScarlet reporter, luciferase reporter, and neomycin resistance reporter. Image...hypothesis with GFP fluorescence. To do this they generated a silent GFP construct, that contains an ACG start... of different reporters including mScarlet-I, luciferase, antibiotic resistance markers, and other enzymes...fluorescent proteins, antibiotic resistance, and luciferase. References Gaudelli NM, Komor AC, Rees HA,...CRISPR/Cas9-mediated Genome Editing. Molecular Therapy - Nucleic Acids 5:e334 . https://doi.org/10.1038...

... the ASM General Meeting (now Microbe) in 2013 in Denver, CO. While I had been to the General Meeting ...the next generation of scientists. The connection between mentors and Singletons can accelerate the networking...meet someone new, as they’d been introduced to several people by their mentor, as well as their bin-mates...recognized by meeting organizers in their town hall.  Iterative improvement - Making Binning Singletons better...Goldschmidt conference, Coastal & Estuarine Research Federation (CERF) Biennial Conference, Gordon Research Conference...

...process to reduce viral order fulfilment from an average of 40 minutes to 10 minutes. While users can’t ...result in less than optimal user experience and the overall soundness of the software design. Each of us brings...called Scrum. The Scrum framework is based on an iterative and collaborative approach to development. Scrum...capacity to finish all of the work. Since we work in iterative chunks, we have the flexibility to adapt and easily...allows for life to happen without impacting the overall work. Each of our sprints is themed and the stories... being maintained (i.e. hasn’t been updated in several years) or they make significant changes that make...that helps our users focus on the fun stuff, accelerating research and discovery. We work with transparency...

...molecule into the cell. The fluorescence that is generated allows for qualitative and quantitative measurement...contact with its RNA aptamer. This increased interaction between Corn and its small molecule makes Corn...developed to quantify RNA transcription by RNA Polymerase III. By expressing Corn under promoters for transcripts... transcripts uniquely transcribed by RNA Polymerase III (5S, tRNA and U6) and measuring Corn fluorescence...effect of mTOR inhibition on the activity of RNA Polymerase III. Red and orange fluorescent aptamers Corn...changes in intracellular viral rna." Journal of General Virology 98.5 (2017): 992-1003. PubMed PMID: 28555544...PMC5602116. Song, Wenjiao, et al. "Imaging RNA polymerase III transcription using a photostable RNA–fluorophore...

...of organelles and vesicles, and more. Although several strategies are available to disrupt microtubules...extended to other GTPases. The Chow Lab has also generated a variety of membrane-recruitment optogenetic ...recently developed a full toolbox and workflow for generating new Rho GTPase optogenetic systems (Berlew et...express the other separately, then use blue light to generate CRY2-CIB1 oligomers that trap the fusion protein..., and 100 times the activity of the original, generated using directed evolution (Tei et al., 2023).  ...license.   Just make sure you don’t overdo it! The moderately-enhanced 10x and 30x SuperPLDs avoid dark-state...Krishnan, N., Ononiwu, F., Pal, D., Manikas, J., Taveras, C., Hall, N. A., Silva, J. D., Freshour, J., &...

...these techniques are cumbersome, often requiring several rounds of selection to isolate plants with the ...organisms like Arabidopsis and Medicago truncatula and several crop species including potato, corn, tomato, wheat... plant cells.   This blog post will present a general overview of plant genome engineering using CRISPR... repress target genes in plants using the same general experimental design principles developed in other...systems, multiple gRNAs can be expressed to modify several genomic loci at once (get more information on multiplexing...sequence to enhance targeting to the nucleus, and several codon optimized Cas9 variants have been created... the plasmids described above, Addgene carries several useful CRISPR toolkits for creating plant expression...

...highly multiplexed tissue imaging method that uses iterative staining and chemical bleaching to allow for high-resolution...high-quality antibodies and reagents that you know work…preferably before you start your experiment. (And you’re...the IBEX Knowledge-Base, seeking to create an iterative process that is constantly becoming better, with...Ichise, H., Gola, A., Kabat, J., Lowekamp, B., Speranza, E., Croteau, J., Thakur, N., Jonigk, D., Davis...Hernandez, J. M., & Germain, R. N. (2022). IBEX: An iterative immunolabeling and chemical bleaching method for...00644-9 Radtke, A. J., Kandov, E., Lowekamp, B., Speranza, E., Chu, C. J., Gola, A., Thakur, N., Shih, R...

...to three) working in your chosen field. This cooperative education experience (co-op) is what has brought...find out that the two hours were split up between several Addgene employees, who all made the daunting task...old) employees with information on how Addgene operates as a team and shows how they fit into the system... on projects there. At this point, I have seen several lab processes and am currently working on projects...are scheduled to take place and employees are generally expected to be available. In addition to these...student at Northwestern University. She is one of several students completing their co-op at Addgene. This...

... to the molecule it detects. This change then generates a reporter signal. Reporter signals may be electrochemical...fluoresce; when present, calcium induces CaM/RS20 interaction, thereby changing the FP’s structural organization...M13. When calcium binds, it triggers CaM/M13 interaction that increases FRET transfer between the fluorescent..., and voltage across membranes. These sensors generally follow the same pattern described above - a sensing...withstand a particular solute concentration or temperature? For further advice, see our posts on how to ... Plate-based assays (plate reader) Detect the average amount of fluorescence across a population of cells...

...the relevant literature to understand where the field is today? Have you talked to veterans in the lab ...-to-date with the literature of a couple of well-known journals that discuss general science in the area... timeline? A typical research project can take several years to complete and has multiple benchmarks. ...before even touching a pipetteman. Take the time to generate new ideas and think about them critically before...

...overlapping or nested on a register, then the operation of one recombinase on the register could affect...affect the way downstream recombinases interact with the register- either by re-orienting their recognition...amplified by a particular primer pair. Then, by leveraging qPCR data from multiple primer pairs, one can...designed to have 20-30 nt length, ~60 oC annealing temperature, and ~50% GC content) so that individual parts...not – the state machine design space is highly degenerate and often times there are multiple register designs...serine recombinases have been described in the literature (5, 6) and may be substituted for one another...predictability of RSMs and recombinase-based circuits in general would benefit from a more comprehensive characterization...

...looking to determine how Protein A and Protein B interact. You read extensive research on the two proteins... both targets in your specimen. You scour the literature and find an ideal antibody against Protein A ...reduce immunogenicity and thereby enhance the therapeutic potential of antibody medicines. In this process...with human antibody sequences.  Additional considerations Although isotype conversion is routinely used...utility of recombinant antibody tools, the resulting chimera may have different binding properties (Morelock...needs to be addressed when multiplexing. It is imperative that you use narrowly selective isotype-specific...

...previously yielded valuable enzymes. Famously, Taq polymerase – from Thermus aquaticus bacterium – was discovered...processing environmental samples Since the high temperatures and unstable ground around these hot springs...collection site, we took GPS coordinates, water temperature readings using a heat gun, and pH measurements... continued profiling samples, and years later, several whole genome sequencing projects were launched ...Parvibaculum lavamentivorans, whose optimal growth temperature is 30°C. Find IgnaviCas9 here! What can you do...applications where reagents must be stored at elevated temperatures for long time periods and workflows where CRISPR...

...Thailand, our annual operating budget is approximately 20,000 USD and we are working on several genetic diseases...: This year, the BeHEARD (Helping Empower and Accelerate Research Discoveries) Award attracted submissions...affected by AHC suffer from recurrent attacks of unilateral paralysis (hemiplegia) that are often painful...stressors, including emotional stress or changes in temperature.  “The disease was first described in 1971,” ...elucidating association between SOX10 and WS4 and also several other rare genetic diseases. We hope kids in Thailand...

...outcrossed knock-in ready for experimentation. In 2014, several manuscripts published in Genetics detailed approaches... DNA as repair templates which reduces time to generate edits to only four days (Paix et al., 2017). The... demonstrated that selection for repair of a temperature-sensitive pha-1 mutation significantly enriches...design The Meyer lab meticulously characterized several aspects of editing. First, they devised guidelines...modifying the 5’ modification of oligos used to generate linear dsDNA repair templates by PCR boosted efficiency...from a single injected animal.  PAM sequence considerations For changes 5’ to the PAM, sequences corresponding...elegans The Meyer and Mello labs also identified several experimental set ups that could improve CRISPR ...

...development of useful diagnostic methods and improved therapies for PZA-resistant Mycobacteria tuberculosis. Investigation...to tag individual strains of bacteria. Their interactions and growth within the biofilms will then be ... taraxacifolia leaves are widely used to treat several diseases. Her goal is to identify and isolate pure...Therefore, understanding neuronal networks, their interactions and information processing during this condition... to get new insights into functional network interactions. Combining synthetic biology with engineering...resources and microfabrication technologies to generate a self renewable sensor, a bacterium that can ...

...remote collaborations Transferable Skills Guide: External collaborations Transferable Skills Guide: Teamwork...how to seek out such an arrangement, and how to operate once in that position. What is a visiting scientist...new placement. Make sure you read any relevant literature for the techniques you will be performing to ...home institute. Discuss rights Do you expect to generate data that will contribute to a publication? A ...authorship and rights for anything that could be generated during the visit. If you don’t feel comfortable...