...Belarusian State University Series of Physiological, Biochemical and Molecular Biology Sciences. 9.1 (...genomic modification. This technique is frequently called allelic exchange. Here, a mutated allele introduced...: The initial plasmid integration step (the so-called “single crossover”) which results in a merodiploid...They hypothesized that genes that had evolved specifically for toxicity would allow stronger counter-selection...system is well-characterized and quite tight (Giacalone et al., 2006). The Chen lab demonstrated excellent...synthetic ribosome binding site (using the online calculator from the Salis group) (Espah Borujeni et al.,...from TP997). We validated these vectors, which we call “pTOX” in a broad group of Gram-negative Enterobacteriaceae...

.... Lajoie, Marc J., et al. "Genomically recoded organisms expand biological functions." Science 342.6156...phosphorylation events on the activity of a given protein? Classical methods for studying protein phosphorylation A...plasmid. This includes 4 copies of a tRNA that specifically recognizes amber stop codons (TGA) to encode...effects on cell growth. The C321.ΔA strain was genomically recoded to eliminate all 321 amber stop codons...strain, TAG is an ‘open’ codon, which can be specifically recognized by the Sep-tRNA. 3. An expression...specific phosphoserine residue at this position. Practical tips for your experiments To generate as much ...containing Phos-Tag, a small molecule that binds specifically to phosphate moieties and promotes a robust ...

...methods including both chemical and physical methods in the lab. Chemicals like calcium phosphate and diethylaminoehtyl...competent through chemical manipulation in the lab. This is commonly done using calcium chloride which permeabilizes...also use electroporation, the application of an electrical charge to cells, to increase cell membrane permeability...contact. The donor bacterium contains a DNA sequence called the Fertility factor (F-factor). The F-factor is...easily cross the negatively charged cell membrane. Physical methods such as electroporation or microinjection...cells. Electroporation on the other hand uses electrical pulses to create transient pores in the cell ...

...almost always epigenetically silenced, but dysregulation of these systems can lead to local dsRNA production... DNA – right? RNA performs almost all of its biological functions in our cells in the single strand form...machinery of the host. This pathway has been clinically exploited for antiviral purposes similar to that...sequence. Once a compatible sequence is found (typically RNA that the RNAi is designed against), RISC can...target mRNA. These RNA tools have been used in clinical therapy to target pathogenic proteins, as well...and insecticides, an advancement over previous chemical versions with toxicity to many organisms. ...transcription of their circularized DNA. These dsRNAs are typically contained within the mitochondria, barring a loss...

...fluorescent genetically encoded voltage indicator Article contributed by Shreya Vedantam Genetically encoded... for its biotechnological applications including conversion of CO2 into water and electrical energy generation...can be reconstituted in vivo to form a protein identical in sequence to the unmodified enzyme. Each half...tested the base editors in vivo and achieved therapeutically relevant editing efficiencies in the mouse ... base editors at Addgene. Levy et al., Nature Biomedical Engineering, 2020. PubMed PMID: 31937940. Detection...single action potentials and further studies of optical neural circuit manipulations when combined with...fusion proteins separate, inactivating Cre. This is critical for reducing Cre toxicity in long-term experiments...

...detect mRNA from 10 bacteria of clinical significance and four clinically relevant host biomarkers. The ...microbiome with sequencing is vast, the ability to genetically alter microbiome members is limited and many ...at Columbia University developed a method for genetically altering gut microbiome members in their native...native habitat (Ronda et al., 2019). They called their method Metagenomic Alteration of Gut microbiome by... analyze gut microbiota and host biomarkers In clinical and diagnostic labs, deep sequencing and qPCR ...large number of microbiome samples. To enable large-scale, longitudinal studies of the gut microbiome, James...The lab validated their platform on clinical stool samples by comparison to qPCR and demonstrated that it...

...ability to specifically recognize and bind proteins. They’re used in a number of research and clinical applications...Instead, we're going to focus on what I'm going to call "alternative affinity reagents" - everything from... of the antibody’s ‘Y’. They are often used in clinical applications. Fab fragments can be created in ...other (Shahied, 2004). They are often used for clinical applications, including imaging.  Diabodies are... from a camelid antibody lacking a light chain, called a heavy chain antibody (Muyldermans S., 2013). ...Designed ankyrin repeat proteins, or DARPins, are genetically engineered small affinity proteins, around 14...proteins known as ankyrin repeat proteins. DARPins typically consist of 3-4 repeats, have high specificity ...

... the practical aspects of testing samples in the lab, but I really struggled with the large scale data... this summer position as I felt I needed more practical lab experience to later gain a full time position...really enjoyed some aspects of the work, such as physically carrying out experiments like PCR and running...least 3 months experience working in a developing tropical country. This is why I set up a placement working...first aid. This really allowed me to see the practicalities of working in the field with the most basic...Masters, because tuition fees had just gone up dramatically and, in England, you could not receive government...carry out my dissertation research in a lab in California. I felt then, and still feel now, that it is ...

...accurately calculate the protein concentration of any experimental wells in that range of optical densities.... When an ELISA kit is ordered, the wells are typically already coated with the antibody. Then, you add...process is the addition of a detection antibody (typically a monoclonal antibody) that also recognizes the... of enzyme present in the well and is used to calculate the amount of your protein of interest that is...is present. Using a Standard Curve To do that calculation, you’ll need to include a standard curve in your...concentrations of your protein of interest. The optical density (OD) - the measure of light absorbed by...samples can then be plotted along this line to calculate the protein concentration. Image from Jagarlamudi...

...et. al.). Physical versus functional titer Viral titers usually come in two flavors, physical or functional...the target cell’s genome. Physical titer The two most common assays for physical titer are direct p24 measurements...functional. Physical titers measure the amount of viral particles in a sample and are usually based on the...produced can actually infect cells. These assays typically involve infecting a target cell line with your...methods tend to be more accurate than measuring physical titer, there is a risk that they may underestimate...target gene. Developing the proper control line typically requires transducing with a limiting multiplicity...will the target cell type used. Therefore, it is critical that standard conditions be used when comparing...

...protein expression for experimental, biotechnological, and medical applications. Researches can visualize...of proteins including antibodies and pharmaceuticals, specifically interleukins. Yeast expression systems...tagging them with fluorescent proteins to study localization or purify proteins to study their structure,...these types of insect expression systems can be scaled up for production of large amounts of protein. ...time consuming as plant tissues themselves are biochemically complex. To circumvent these issues, scientists... taken advantage of the natural secretion of biochemicals and proteins through plant roots. Tagging recombinant...easily adapted to fermentation allowing for large-scale, stable production of proteins. In general yeast...

... curve to calculate its experimental value. Though it varies from assay to assay, typically the experimental...before running a western blot. Standard curves are critical for a number of popular scientific applications...curve by serially diluting a known control sample, called a standard. The ideal standard curve has at least...enzymatic assays are sigmoidal. Once the trendline is calculated, you can extrapolate the concentration of the...measurements and the trendline y = 0.6561x + 0.019 calculated. The concentration of an unknown sample is determined...standard curve scientists use graphing software to calculate the data’s coefficient of determination, R2. R2...running assays that rely on a standard curve, it is critical that the unknown sample’s concentration lies within...

...COVID-19 cases each week. Statistical Modeling Note -- If statistical modeling isn’t up your alley,... of Chemistry and Chemical Biology at Harvard University; and Howard Hughes Medical Institute Investigator...as expected, plasmid orders (left, blue) drop dramatically when there are many new COVID-19 cases being...order several plasmids at once, breaking the statistical independence assumption of the Poisson process...our distribution, resulting in the following hierarchical model: Because the U.S., Europe, and China ...multivariate Gauss-Hermite quadrature, since the analytical expression for the data likelihood contains an...His research uses applied machine learning and statistical methods for fundamental scientific discovery ...

... general public. Chemical Society in University College Dublin, Ireland The Chemical Society was founded...way up to professors. Oxford Biological Society, England The Biological Society based at the University...idea of the Freshers Fair and some of the great biological societies that Addgene has had the opportunity...at Freshers Fairs in the past year. Cambridge Biological Society, England Since 1996, Cambridge Biosoc...Biosoc’s goal has been to promote interest in the biological sciences amongst the student community at the...At Addgene, we’re incredibly glad to see these biological societies engaging with young scholars. We believe... to keep up the good work! What scientific or biological societies were you a part of in undergrad or ...

...graduate students and postdocs to think analytically and critically. Passion fuels the drive to continue ... Connie Cepko (Harvard Medical School) , and Dr. George Church (Harvard Medical School) for taking the...people (especially you!) care about. Have you critically evaluated your plan? Have you read the relevant...write a grant for this project, what would be your critical experiments and why? What techniques and skills...help move your project forward? Most projects typically require a diverse set of skills. It is important...direction of the project? What is your timeline? A typical research project can take several years to complete...time to generate new ideas and think about them critically before diving in. Research projects are designed...

...of Various Motifs is Critical for Initiation of Replication. Journal of Biological Chemistry, 273(14), ... replication machinery. For instance, the ori typically contains iterons that serve as recognition sites...Acinetobacter spp., Aeromonas spp., Agrobacterium spp., Alcaligenes spp., Aliivibrio salmonicida, Anabaena spp., ...Gram-negative and Gram-positive  Acinetobacter calcoaceticus, Actinobacillus pleuropneumoniae, Actinomyces...Aeromonas hydrophila, Agrobacterium tumefaciens, Alcaligenes eutrophus, Azotobacter vinelandii, Brevibacterium...pSa, pR388 Gram-negative  Acinetobacter calcoaceticus, Aeromonas liquefaciens, A. salmonicida, Agrobacterium...Agrobacterium tumefaciens, A. rhizogenes, Alcaligenes eutrophus, Enterobacter sp., Erwinia amylovora, E...

...tricks By Ashley Waldron While IHC protocols typically call for thin sections of tissue, the Wu Lab is ...therapy, Feng Zhang’s lab has developed a system called DIRECTED (Delivery to Intended REcipient Cells ...promising approach for experimental therapies and medical applications.  Find DIRECTED plasmids here! Strebinger...labeling can identify protein interactions or map a local proteome, but some applications have been challenging...control over when and where the enzyme is active, drastically reducing background labeling caused by the biotin...proximity labeling enzymes, TurboID and APEX2, are localized to different cellular compartments. They showed...of a cell. Lin et al. generated a fusion protein called iPAK4 that assembles into a slow-growing protein...

...connections to that population. Limitations of common genetically encoded neuronal tracers Neuronal tracers such...neurons. As compared to epitopes tags, FPs are intrinsically fluorescent and can therefore be used in live...tolerated by cells. They can be used for protein localization, isolation, and tracking experiments (Rizzo,.... Thus they have created antigenic protein tags called “spaghetti monster” fluorescent proteins (smFPs.... Indeed, to create the smFPs Looger’s team strategically inserted 10 to 15 copies of single epitope tags...darkened chromophores. These probes are both intrinsically fluorescent and can be detected with highly ...postsynaptic cell adhesion protein that plays a critical role in neural development. It belongs to a huge...

...The good news is that viruses are very valuable biological tools, and with the right safety considerations...infiltration into a cell. The twist is that the typical genetic cargo — the viral genome — is swapped, ...vector containing the cargo of interest is then typically transfected along with viral packaging plasmids...risk management when working with viral vectors. Typically, one day after transfection into the production...expression or integration resulting from transduction typically plateaus within a few days to a few weeks, depending...context you are targeting? Second, what is the biological outcome you want to achieve? For example, do ...swap out your virus type to achieve different biological outcomes. For example, some viruses integrate...

.... Factor Xa loves calcium, but metal-chelate matrices, not so much. Maybe a calcium-independent proteinase...This allows washing away of unbound material to calculate the amount of bound ligand (after subtracting ...subtracting out non-specific binding). Historically, glutathione-S-transferase (GST) fusion proteins have been immobilized...derivatives streptavidin and neutravidin) is for all practical purposes covalent. Therefore, protein immobilization... four biotin-binding sites per molecule so, theoretically, saturated avidin could form tetramers of biotinylated...well-controlled. The lack of site selectivity typically diminishes the apparent affinity for the ligand...However, large amounts of the target protein are typically required due to the relatively low efficiency ...