...Golden gate assembly limitations Embraced by the synthetic biology community, Golden Gate Assembly is commonly...in-frame assembly proceeding in 99% of 12-fragment assemblies and over 90% for 24-fragment assemblies (Figure...any Golden Gate assembly design. Number of fragments assembled Golden Gate Assembly protocol* (Volume...from 2 µl of the assembly reaction, and from the entire assembly reaction. All assembly reactions had a... 20+ fragment assemblies with both robust efficiency and accuracy. Golden Gate assembly involves the ...enhancing the ordered assembly of the target construct. Development of Golden Gate Assembly test systems Our...different levels of assembly: High efficiency and accurate single-insert assembly Intermediate 5- or ...

... generated a fusion protein called iPAK4 that assembles into a slow-growing protein fiber in the cytoplasm...points appear as fluorescent bands in the fiber, resembling a “ticker tape” recording. The authors created...

...able to come of age. The in vivo assembly cloning protocol In Vivo Assembly (IVA) cloning (Garcia-Nafria ...recombination events to assemble the final plasmid. At least 5 fragments can be correctly assembled using IVA cloning...majority of current cloning techniques involve the assembly of a circular plasmid in vitro, before transforming...propagation. However, while not widely known, plasmid assembly can be achieved in vivo using a bacterial recombination... generation - Any linear DNA fragments can be assembled in vivo as long as they have homologous sequences...fragments are transformed into E. coli and plasmid assembly (re-circularization) occurs in vivo. We use XL...insertions, deletions, and point mutagenesis to complex assembly of multiple genes. Primer design for each type...

...of DNA assembly available, including: Gibson Assembly®, BioBrick® Assembly, Golden Gate Assembly, and NEBuilder... in DNA assembly will still yield scarless assemblies. Overview of NEBuilder HiFi DNA assembly method ...England Biolabs. What is DNA assembly? In the context of cloning, DNA assembly refers to a method of physically... NEBuilder® HiFi DNA Assembly. Gibson Assembly allows the production of scarless DNA constructs using ...enzymes results in scarless assembly. Finally, the NEBuilder® HiFi DNA Assembly method from New England Biolabs...planning an NEBuilder HiFi DNA Assembly mix reaction is the NEBuilder Assembly Tool which is freely available...complicated assembly approaches, and opens the door for new opportunities in the field.  Gibson Assembly® is ...

...60,000 views for the month of September! Historically we do better in September than in the summer months,...tired of calculating dilutions or you can’t quite remember the hazards of that chemical in your obscure growth... an added bonus. You can find the rest of our September posts in chronological order here: Four Things...hoping that October is even more awesome than September! Tyler J. Ford ...

...This year, Addgene welcomes three new members to the Board of Directors: Khandan Baradaran, Frank Stegmeier...set of experiences. Let’s meet the newest board members and get to know them! Khandan Baradaran, PhD Khandan...the Ocular Genomics Institute, and an Associate Member at the Broad Institute of Harvard and MIT. ...

...structure of CasX at eight different states of assembly from cryo-electron microscopy, the team reveal...

...with proteins and remain functional, 2) break membranes outside of their natural context or 3) be modified...demonstrates that R bodies can function to break membranes of different organisms. And finally, we demonstrated...could use them to lyse cells (and surrounding membranes) in keeping with their natural function (see below...). With a better understanding of how R bodies assemble and function, we might be able to endow cells ...expression of genetic determinants for synthesis and assembly of type 51 R bodies. J. Bacteriol. 168, 40–48 ...Silver, P. A. A Tunable Protein Piston That Breaks Membranes to Release Encapsulated Cargo. ACS Synth. Biol... membrane...

...perspectives. Such activities foster a mindset that embraces the unexpected and encourages exploration of tangents...ideas and approaches. Moreover, the ability to embrace serendipity fosters adaptability, a crucial skill...groundbreaking discoveries. So, on your academic journey, remember to leave a little space for the unexpected — it...

...specific area at the plasma membrane (Figure 2). Just express the plasma membrane-localized iLID along with...cell membrane isn’t powerful enough, what about altering the chemical composition of the membrane itself...SuperPLD hydrolyzes membrane PC to PA. A) OptoPLD is recruited to plasma membrane or lysosomes and its...optogenetic tools to control the cytoskeleton and cell membrane dynamics. First, a quick refresher.  The tools...undergoes a conformational change and binds the plasma membrane under blue light CRY2-CIB1 heterodimerizes or ...new optogenetic tool for localized microtubule disassembly, Opto-katanin (Meiring et al., 2022), that overcomes.... The Chow Lab has also generated a variety of membrane-recruitment optogenetic tools for manipulating...

...sybodies against membrane proteins Developing antibodies against proteins embedded in membranes is tricky because...to 1012 members) in comparison to other display systems using phage and yeast (109-1010 members). This ...develop antibodies against unstable targets like membrane proteins that may unfold due to camelid high body...synthetic nanobodies, called sybodies, against membrane protein targets in as little as 3 weeks (Zimmermann...lab can quickly select for sybodies targeting a membrane protein of interest using the Sybody Generation... the process for selection of sybodies against membrane proteins. Image from Zimmermann et al., 2018. ...SARS-CoV-2 Receptor Binding Domain found on the membrane-bound spike protein, in a remarkable 12 working...

...We’re happy to introduce the newest member on Addgene’s Board of Directors, Bart Newland. Newland is ...does your experience help you as an Addgene board member? BN: Having seen so much in the business world ...

...insight to the now 13 member board at Addgene. Let’s meet the newest board member and get to know her better...Addgene from Joanna Brownstein, an Addgene board member. When I was working in Human Resources at Harvard...prior work experiences help you as an Addgene board member? It’s not like I understand the science but my ...advance the mission of the school. She was also a member of the Northeast Human Resources Association, Association...blog Resources on addgene.org: Meet the other members of Addgene's Board of Directors Find careers at...

...circular assemblies. While Golden Gate Assembly worked for all the assembly types, circular assemblies yielded... circular assembly of the T7 genome had lower efficiency than the 10-piece circular assembly, with the...accomplished with Golden Gate Assembly—even when getting help from DAD. The 35-piece assembly mentioned above had...limit for Golden Gate Assembly size. Ultimately, data-driven Golden Gate Assembly greatly increases the...complex Golden Gate Assembly, a great place to start is the 24-piece lac operon assembly deposited by NEB...-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design. PLOS ONE...If you’ve ever used Golden Gate Assembly for cloning, you might be familiar with the rules of thumb for...

...insight to the now 10 member board at Addgene. Let’s meet the newest board members and get to know them...looking for new board members. Paratore: Kristen Darby, who’s also a board member, reached out and asked...Dorr and Crowell & Morning. Karen was a founding member of Mass Medical Angels, a venture mentor at MIT...certified health insurance executive (CHIE). He is a member of both the Internal Institute of Auditors and ...blog Resources on Addgene.org Meet the other members of Addgene's Board of Directors Find careers at...

...basics of Gibson assembly as a parody of “Breakfast at Tiffany’s.” Overview of Gibson assembly The Gibson assembly...gain in popularity. Gibson assembly is well known for allowing easy assembly of multiple linear DNA fragments...Miller Lab Protocol). The Gibson assembly process can be used to assemble up to 6 fragments in one step,...design primers for Gibson Assembly. For a simple example of using Gibson assembly, imagine that you want ...annealing and priming of neighboring assembly fragments. Gibson assembly is commonly used in synthetic biology...in 2013 found that Gibson assembly was one of the most commonly used assembly methods (Kahl 2013). However...cut out via CRISPR and then assembled into a vector using Gibson assembly. In this case the vector was...

...to different lipid compositions of C. elegans membranes, which might beneficially influence the rhodopsin...

...podcast segment A fundamental approach to studying membranes, organelles, and other subcellular compartments...

...started, the hardest part of the process was to embed personality in pieces of plastic, but it’s gotten...pictures, one for each position in a movement, and assembling the many pictures together to form the full movement...when we started was creating a LEGO stage that resembles a real laboratory. It only took one night to build...

... different barcodes to each of the gRNAs and to embed these barcodes within the gRNA sequences. This allowed...

...fluorescent voltage indicator. Archon1 can embed itself into the cell membrane where cell voltage can then be measured...

... your membrane, run a double-membrane transfer with a psi membrane stacked behind the p membrane. If your...through the p membrane into the psi membrane, that’s a good indication that the p membrane pores are too...marked on the membrane (1) using the gel as a guide. The gel is then removed from the membrane (2) and the...troubleshoot.   Before you start… Mark your membrane! Membranes look the same from both sides, so it’s quite...appropriately sensitive. If using a PVDF membrane, you can dry the membrane, wet it with 20% methanol, and place...completely through the membrane or larger proteins not transferring completely onto the membrane. If you’re not...completely through your membrane to the other side), you can check by using two membranes during your transfer...

... your gel and membrane transfer as normal. Immediately after transfer, dry the membrane to maximize protein...manufacturer’s instructions for the membrane you are using. Dry nitrocellulose membranes are brittle, so proceed ... as normal! Stripping your membrane Alright, so you’ve dried your membrane and returned it to buffer, ...depend on your specific experiment. Remember, stripping your membrane will remove some of your sample —and...’ll want to: Rinse the membrane in water or fresh buffer. Cover the membrane in stripping solution and...a fume hood. Rinse the membrane in water or fresh buffer. Cover the membrane in stripping solution and...you need to start a whole new blot? Never fear: membrane stripping is here! Rather than start over, you...

...off-target editing sites in the embryos, as well as whole exome sequencing of embryo derived-ES cells.They also...editing between iPS cells and embryos make a case for conducting research in embryos should our society decide...sequencing to a small percentage of edited embryos. Since the embryos produced were not being used to establish...-free embryos to only 60%? Unfortunately, given the differences between CRISPR repair in embryos and iPS...this paper. Any hint of CRISPR editing in human embryos has been met with a storm of media coverage. But... replication. Indeed, they observed that 41/42 embryos were non-mosaic at 3 days post fertilization. Since...MYBPC3 allele. Interestingly, they found that the embryos underwent repair not from the single-stranded oligonucleotide...

...nitrocellulose membranes and exposed to IPTG in order to induce protein expression. Cells on the membranes will ...orientation of each membrane on its plate before moving on. Then peel each membrane off the plate (saving...high salt.  To probe membranes with target bacterial cells, wash your membranes with PBST to remove salt... on the membrane to bind their target cells, pulling them out of solution. Wash your membranes well with... 20 Petri dish-sized circles of nitrocellulose membrane and press each gently down onto a plate of colonies...regrow the lifted colonies overnight), flip the membrane over and place it colony-side up onto a fresh ...plasmid selection. Remove air bubbles between the membrane and the agar using forceps. Several protein induction...

...multicomponent assembly, as fragments with overlapping sequence homology can be assembled in a unique order...multicomponent assembly have 5′ or 3′ sequence homology to each other, they may be assembled incorrectly.... one option is to perform a hierarchical assembly, assembling fragments in multiple steps to avoid using...Golden Gate assembly, may also be beneficial. SLIC is most often compared to Gibson assembly, a cloning...required for Gibson assembly. SLIC is a standardized method for multi-fragment DNA assembly, and its low cost... by Mary Gearing in December 2015 and was updated by Emily P. Bentley in September 2024.  References ... cheap, standardized, and rapid multi-part DNA assembly method. Read on to learn how to use it in your...

...to use some assembly methods such as Golden Gate or Gibson assembly. Golden Gate Assembly of gRNAs Golden...Golden Gate/Gibson Assembly Multiplexing Plasmids: These plasmids allow you to assemble 2-4 gRNAs through...gRNAs. PTGs are assembled into a wt Cas9-containing vector using Golden Gate assembly, and up to 8 gRNAs...Multiplex Plasmid: Two gRNAs can be assembled using Gibson Assembly or SLIC cloning. gRNAs are expressed...Bullock lab, and gRNAs can be inserted using Gibson Assembly or SLIC cloning methods. A BsaI-based E. coli ... multiplexed gRNAs with Golden Gate and Gibson assembly methods If you want to scale up the number of ...Golden Gate Assembly uses Type IIS restriction enzymes, which cleave outside of their recognition sequence...

...can be used to assemble multiple fragments - up to 10 fragments are commonly assembled in a single reaction... Gibson assembly require 20-40 bp of homology at the ends of DNA fragments to specify assembly order, ...sequence homology cannot be assembled using this method, but can be assembled with Golden Gate. The popular...acids, but Golden Gate assembly can be designed to be scarless. Golden Gate assembly is also less expensive...recognition sequence, they can be customized to direct assembly of DNA fragments. When designed correctly, the...sites with complementary overhangs that direct assembly of the final ligation product. As shown below,...higher number of potential clones. Golden Gate assembly has a few advantages over other cloning methods...

...organization scheme for multi-part DNA assembly. Simply put, it assembles individual DNA components into larger...MoClo-compatible vectors. Level 1: Assembly of multiple Level 0 parts into TUs. Level 2: Assembly of multiple Level 1...IIS restriction enzyme and a ligase are used to assemble multiple DNA fragments together in a one-tube ... Fig 1. MoClo enables individual parts to be assembled into larger functional units which are transcribed... Understanding how individual parts can be assembled into TUs is key to understanding MoClo. By a similar...Construction of multigene constructs, through multi-TU assembly,happens via three steps: Level 0: Cloning of ...multigene constructs. Figure 2: The three assembly levels of MoClo. Image courtesy of Rob Hurt. ...

...modules. In the first assembly step, compatible Level 0 vectors are directionally assembled into a Level 1 vector...of Level 1 assembly if necessary. Combining multiple Level 2 vectors in the final assembly step permits...structural and spectral properties of two of the family members – one with an unprecedentedly-high brightness, ...relies on several observations: Rapamycin is a membrane-permeable pharmacological agent that can rapidly...extraneous proteins bound to its outer mitochondrial membrane. Sequestration is accomplished by knocking down...protein with a FKBP domain and a mitochondrial outer membrane protein with a FRB domain (MitoTRAP). Rapamycin...., J Cell Sci 2013 May 1;126(Pt 9):2102-13. Assemble multiple component plasmids from building blocks...

... cells in an organism, including the embryonic and extraembryonic cell types. In mammalian development...generation does not require embryos, their use skirts issues surrounding the use of embryos in research. With all...Beckmann, and W. Wurfel, Non-embryo-destructive Extraction of Pluripotent Embryonic Stem Cells: Implications...-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic...totipotent cells. An example of pluripotent cells is embryonic stem cells (ESCs), which I discuss in detail below...into two broad types according to their source: Embryonic stem cells ESCs are pluripotent stem cells that... abnormal and/or supernumerary preimplantation embryos that are produced during in vitro fertilization...

... PI helps lab members determine project "ownership" and author order PI lets lab members fight it out ...long list of written questions for the PI and lab members.  Make coffee or beer dates to talk to as many ... informative way of gathering data. Former lab members can give open, revealing information about the ...and lab head. While it’s good to talk to new lab members too, they may not have had experience writing, ...career opportunities with the lab head. Former lab members can give you a fuller picture. Consider talking...try to attend a lab meeting. Observe how the lab members treat one another (especially those low on the ...will work for you, ask questions like: Do lab members feel there are enough projects to go around? What...

...zebrafish embryo develops outside of the mother, making them ideal for studying vertebrate embryogenesis. To...investigations into environmental pollutants and health, embryo growth, brain function, and disease development...illustrate some of the reasons that zebrafish have been embraced as a model organism, I’ll outline a few of the...quality genome sequence A high-quality sequence assembly of the zebrafish genome was published in 2013,... when and where genes are active in developing embryos are available. Additional analysis tools have been...head, eyes, and tail. Compared to the opaque mouse embryo, where similar processes occur over days inside...the zebrafish is an ideal system to observe embryogenesis live. The origins, divisions, and movements ...

...method. Membranes The two membrane options are PVDF and nitrocellulose. PVDF is a sturdy membrane that can...low-background PVDF membrane, it also has higher background noise than nitrocellulose membranes. Pro tip! You...visualize proteins on a PVDF membrane without staining; simply wet your membrane with 20% methanol and place...designing the experiment. What detection method? What membrane? What should you block with? It can be so overwhelming... location of your protein. Proteins located in membrane-bound subcompartments, like the mitochondria or... protein is difficult to solubilize, such as a membrane-bound protein. There are many different lysis ...the transfer: you’ll need to pre-wet most PVDF membranes before the transfer and allow them to dry afterwards...

... Team, but I’m also an emeritus member of the Scientist Team, a member-by-association of the Development...at Addgene, an unofficial title that I happily embrace (and admittedly, I may have started that nickname...update how we make and distribute kits, I helped assemble a crack team of Addgenies that includes: Michelle...control efforts with depositors; Gizela, the lab member who knows how the physical kits are made and shipped...months, or even a few weeks. The important thing to remember is that every team should have a goal, or at the...and providing technical customer service. Team members are highly trained, and though there are necessary...particular function. Project team A Project Team is assembled to complete a series of tasks with precisely defined...

...and membrane integrity (Fig. 2). For example, saponin interacts with cholesterol in cell membranes and...can lose membrane-associated proteins, but it is better at permeabilizing organellar membranes. Created... work is to try it. As you embark on your next IF adventure, just remember that even though these early...proteins that need a little help crossing cell membranes as they are not able to diffuse across natively...Permeabilization essentially punches holes in the cell membranes of your sample to allow antibodies better access...target is already on the extracellular side of the membrane, then this step may not be necessary. In fact,...and leaves many membrane-associated proteins in place. In contrast, Triton X-100 and Tween-20 are examples...

...Immobilizing proteins on a membrane Once separated, the proteins are immobilized onto a membrane, usually composed...sticky areas on the membrane will bind to the proteins in the buffer, blocking the membrane  from binding to...are: (1) separated by size, (2) transferred to a membrane, and  (3) detected using antibodies.   ...protein is separated in a gel, transferred to a membrane, stained with antibodies, and finally visualized... and high binding capacity. In addition, PVDF membranes can be repeatedly stripped of an antibody and ...follow the same general principle where the gel and membrane are “sandwiched” together with blotting paper ...proteins to migrate out of the gel and attach to the membrane.  In a wet transfer system the sandwich is completely...

...2008 Gibson Cloning Gibson assembly method Single reaction, can assemble large DNA sequences, no restriction...derived from the tyrosine-protein kinase Lyn) Membranes 3 mCherry 587nm/ 610 nm human β-Actin Actin ...same multiple cloning site (MCS). Modules are assembled one at a time by standard restriction enzyme digestion...upon ligation. As a result, once two modules are assembled next to each other, a third module cannot be placed...high-throughput cloning strategies available for assembling these modular types of constructs, and different... Golden Gate Type II restriction enzymes Can assemble up to 10 modules in a single step. Through iteration...restriction enzyme-based). Well suited for the modular assembly of highly similar or repetitive sequences. Intermediate...

...introduced a new assembly cloning platform called GMAP (Gibson assembly-based modular assembly platform). GMAP...Plasmids from:  March (Q1) | June (Q2) | September 2016 (Q3) | December 2016 (Q3) MARCH 2016Enhancer activity...GMAP is based on the Gibson Assembly method, which allows for easy assembly of DNA fragments by utilizing...streamlines the assembly of TALE repeats into a single-tube, 3 day process by utilizing pre-assembled trimers ...literature searching, DNA synthesis, and plasmid assembly. Luckily, researchers from Douglas Densmore’s ...Genomics. 2015. PubMed PMID: 26165496 GMAP - A new assembly platform for generating both simple and complex...overlaps). To expand the capabilities of Gibson assembly and make a more modular platform, the Jacks lab...

...only a small proportion were found on the plasma membrane (top panel in Fig. 1). Of course, this was problematic...DREADD receptors need to be located on the plasma membrane to be activated by the DREADD actuator after it.... Although we found a larger proportion on the membrane in mice neurons, most of them were still cytoplasmic...limited expression of hM4Di‐mCherry at the plasma membrane in monkey neurons, we next hypothesized that a...improve the transport of this DREADD to the plasma membrane. The haemagglutinin (HA) tag is a small 9 amino...resulted in abundant expression of hM4Di in plasma membranes in both species (bottom panel in Fig. 1). Our ...transgene of interest and its localization in the membrane. The lesson we learned from these studies is that...

...following sequencing and assembly. In cases where the QC team suspects an assembly issue, they manually revisit..., de novo assembly in Geneious can still provide additional insights to resolve the assembly and determine... Illumina MiSeq. The raw sequencing reads are assembled into a circularized plasmid sequence de novo (...bioinformatics pipeline developed at Addgene. The assembled plasmid sequence is then compared to the information...confirm:  The size of the plasmid to ensure our assembly result is accurate The gene/insert or any major...additional considerations to resolve. Sequencing and assembly issues Sequence discrepancies are not always a... can arise due to errors in the sequencing and assembly pipeline. There are regions of plasmids that are...

...on the surface, giving the embryo a “spiny and prickly look” that resembled a hedgehog (Nusslein-Volhard...eyes. Scientist thought these thick “eyebrows” resembled those of the comedian Groucho Marx, so the gene...Institute! Sonic Hedgehog, an important regulator of embryonic development The largest conference room at Addgene...hedgehog exhibit abnormal segmentation during embryogenesis. Typically, regularly spaced denticles (bristles...bristles) outline the segments of the Drosophila embryo. In flies with non-functional hedgehog, the denticles...gradient exerting a different effect on the developing embryo depending on its concentration. Genetic mutations...mutant fly ovaries had “squiggly” actin fibers that resemble spaghetti. We have two plasmids in our inventory...

... option: Fragmid. Fragmid is a modular vector assembly process designed for a quick, plug-and-play approach...depositor John Doench reports even the greenest members of his lab, high school interns, were able to use...five days. Did you say… easier? Fragmid’s modular assembly structure takes a lot of the design work out of...  Once you have the components you want, simply assemble them into a destination vector via Golden Gate...Whatever individual components you choose will assemble in the expected module order. (It’s also why you...the Fragmid system by sequencing 60 individual assemblies created over five months. When they tested those...were perfectly correct. Because they created the assemblies in duplicate, they were able to confirm that ...