...Synthetic Biology: Assembly Standards Guide SynBio Resources SynBio Home Browse All SynBio Assembly Standards ...cloning strategies for the assembly of synthetic parts include Gibson assembly and type IIS systems such...Assembly standards define standard prefix and suffix sequences that make it easy to combine components...networks. This is accomplished through the use of assembly standards – combinations of restriction sites ...prefix and suffix sequence at either end of the assembly, even as more parts are added. Therefore, the ...The following table provides information for the assembly standards most commonly used by part registries...primer design. The restriction enzymes used for assembly are the prohibited sites and must be omitted from...

...in an assembly if you want to save time. Working on ice, combine segments in the Gibson Assembly Reaction... Number of fragments assembled simultaneously Multiple fragments can be assembled in one reaction. However...Gibson Assembly (Link opens in a new window) OpenWetWare: Janet Matsen’s guide to Gibson Assembly (Link...Get started with Gibson Assembly Cloning! Protocols... Protocols Gibson Cloning Gibson Assembly Cloning You may also ...Institute developed a novel method for the easy assembly of multiple linear DNA fragments (Gibson et al...three different enzymes, the product of a Gibson Assembly is a fully ligated double-stranded DNA molecule...

...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 ...

...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 ...

... 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...

...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 ...

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

...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. ...

...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...

...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...

...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...

...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...

...subfamily C, member 3 NKG2-E, NKG2E KLRC4 killer cell lectin-like receptor subfamily C, member 4 FLJ17759...subfamily D, member 1 CD94 LGMN legumain AEP, LGMN1, PRSC1 LTA lymphotoxin alpha (TNF superfamily, member 1) LT...family A, member 2 PDA2, PDI, PDIP, PDIR PDIA3 protein disulfide isomerase family A, member 3 ER60, ERp57...FLJ12287, RP35, SEMAB, SEMB SEMA4B sema domain, immunoglobulin domain (Ig), transmembrane domain (TM) and short...MARVEL transmembrane domain containing 2 CKLFSF2, MGC39436 CMTM3 CKLF-like MARVEL transmembrane domain...CKLF-like MARVEL transmembrane domain containing 4 CKLFSF4 CMTM5 CKLF-like MARVEL transmembrane domain containing...MARVEL transmembrane domain containing 7 CKLFSF7, FLJ30992 CMTM8 CKLF-like MARVEL transmembrane domain...

... D, member 1 NR1D2 Plasmids NR1D2, RVR, BD73, EAR-1R nuclear receptor subfamily 1, group D, member 2 NR1H3...Browse the nuclear receptor plasmid collection assembled in collaboration with the Nuclear Receptor Signaling... domain by author. Source: Wikipedia, accessed November 6, 2013. Author: Boghog2 Nuclear receptors are...research labs from around the globe, Addgene has assembled a Nuclear Receptor plasmid collection for the ...NROB1, SRXY2 nuclear receptor subfamily 0, group B, member 1 NR1D1 Plasmids NR1D1, EAR1, hRev, THRA1, THRAL...LXRA, RLD-1 nuclear receptor subfamily 1, group H, member 3 NR2E1 Plasmids NR2E1, RP3-429G5.1, TLL, TLX, ..., XTLL nuclear receptor subfamily 2, group E, member 1 NR2F1 plasmids NR2F1, COUP-TFI, EAR-3, EAR3, ERBAL3...

...PI FusX TALEN Assembly System Contains 336 pFusX clones allowing for Golden Gate assembly of 15-16 RVD ...expression. Feng Zhang LIC TAL Effector Assembly Kit Assembly by ligation-independent cloning (LIC). Validated...plasmids of pre-assembled trimer and tetramer RVD domains designed for rapid TALEN assembly. Kiran Musunuru...expression of Voytas Golden Gate-assembled TALEN, both in microinjected embryos and transfected cells. TALEN...accompanying documentation allow one to efficiently assemble TALEN constructs with custom repeat arrays. TALEN...Bogdanove Joung Lab TAL Effector Engineering Reagents Assembly via serial ligation. Validated in zebrafish somatic...Ekker Platinum Gate TALEN Kit Platinum TALENs assembled by Golden Gate. Validated in several species. ...

...Parkinson's Michael J Fox Foundation MJFF 40427 pEMB017 NP_9409803_LRRK2_1867_2161_L1914N_V2048T_F2083Y...Parkinson's Michael J Fox Foundation MJFF 40499 pEMB018 / LRRK2_BC_1300_2136 LRRK2 His polH Parkinson's...'s Michael J Fox Foundation MJFF 40500 pEMB018 / LRRK2_BC_1300_2148 LRRK2 His polH Parkinson's Michael...Michael J Fox Foundation MJFF 40501 pEMB018 / LRRK2_BC_1300_2170 LRRK2 His polH Parkinson's Michael J Fox Foundation... Foundation MJFF 40502 pEMB018 / LRRK2_BC_1300_2210 LRRK2 His polH Parkinson's Michael J Fox Foundation...Foundation MJFF 40503 pEMB018 / LRRK2_BC_1309_2136 LRRK2 His polH Parkinson's Michael J Fox Foundation MJFF 40504... 40504 pEMB018 / LRRK2_BC_1309_2170 LRRK2 His polH Parkinson's Michael J Fox Foundation MJFF 40505 pEMB018...

...37351 pQC membrane TdTomato IX Membrane Palmitoylation TdTomato Connie Cepko 22479 FUmGW Membrane Palmitoylation...pCAG-mGFP Membrane Palmitoylation sequence from GAP43 EGFP Connie Cepko 61099 Lck-GFP Membrane Palmitoylation...Mitochondria-Outer Membrane TOMM20 mCherry* Michael Davidson 118736 pBOB-CARMIL2 BH domain-GFP Plasma Membrane CARMIL2...protein of interest with known markers. Addgene has assembled a collection of fluorescent fusion proteins that... Michael Davidson 98822 Lck-mTurquoise2 Plasma Membrane Lck N-terminal lipidation signal mTurquoise2 Dorus... Dorus Gadella 98821 Lck-mScarlet-I Plasma Membrane Lck N-terminal lipidation signal mScarlet-I Dorus ... Vamsi Mootha 49153 GFP-Mff Mitochondria-Outer Membrane Mff acGFP1 Gia Voeltz 158007 pCMV-mGold-Mito-N...

...And Ligation) assembly method (Sander et al., Nat Biotechnol. 2011), the REAL-Fast assembly method (Reyon...or REAL-Fast assembly process or lists of plasmid ID numbers required for FLASH assembly. Links to additional... TAL effectors, including designed TALENs, and assembly methods for REAL, REAL-Fast and FLASH Genome...Ligation-based Automatable Solid-phase High-throughput) assembly method (Reyon and Tsai et al., Nat Biotechnol ...Biotechnol 2012). All three of these platforms assemble TAL effector repeat arrays on a single architectural framework...expression plasmids using REAL, REAL-Fast or FLASH assembly. This new version of ZiFiT Targeter enables users...these reagents are provided below: Joung Lab REAL Assembly TALEN Kit Individual REAL TALE Repeat Plasmids...

...separate vectors and then assembled into one construct using Golden Gate assembly. Gersbach MuLE System Mammalian...expression cassettes can be assembled using Golden Gate cloning or the Gibson Assembly. Chen Boosting CRISPR...this Golden Gate and Gateway based assembly method: plasmids for assembly of single gRNA vectors with various...Expression System Description PI Multiplex CRISPR/Cas9 Assembly System Kit Mammalian A system for constructing...-gRNA genes (PTGs) and then PTG cassettes are assembled into a Cas9 expressing vector. Yang A Robust CRISPR...multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/Cas9 vectors in one round...round of cloning by Golden Gate ligation or Gibson Assembly Liu A CRISPR/Cas9 toolbox for multiplexed plant...

...trafficking to the plasma membrane Acute disruption of the synaptic vesicle membrane protein synaptotagmin...measurements of membrane charges A FRET sensor enables quantitative measurements of membrane charges in live...Calcium Calcium sensor in astrocytes (GCaMP6f), membrane-tethered Khakh Lab pZac2.1 gfaABC1D-lck-GCaMP6f...2018 Sep;21(9):1272-1280. Lin Tian Calcium Plasma membrane targeted GCaMP6s calcium sensor A polarized Ca...+) Dynamics in the Mitochondrial Matrix and Intermembrane Space with the GZnP2 Sensor. ACS Chem Biol. ...sensor iATPSnFR1.0 and iATPSnFR1.1 (cytoplasmic, membrane-displayed, expression in neurons, astrocytes) ...reduced ph interference demonstrates augmented transmembrane chloride movement by gerbil prestin (SLC26a5...

...Finger Consortium Originally published in Addgene's December 2011 Newsletter Overview of TALEN Technology Imagine...can be made. Intuitively, one would expect that assembling relatively small, repeat-laden DNA regions into...from the University of Minnesota, arrays can be assembled in a matter of days. In an open access Nucleic...recently developed Golden Gate cloning method to assemble “multiple DNA fragments in an ordered fashion ...custom arrays consisting of 12-31 repeats can be assembled and inserted into a variety of backbones in just... in August, uses a serial ligation protocol to assemble arrays. Dr. Joung, co-founder of the Zinc Finger...early in 2011 and they described their kit for assembling TAL effector arrays in a 2012 issue of Nature...

...pluripotency during embryonic development. This “reprogramming” of the cell to an embryonic state holds immense...cell lineages. Pluripotent stem cells, such as embryonic cells, can potentially give rise to any other ...limited to related cell lineages. Pluripotent embryonic and multipotent adult stem cells have been studied...removes the possible ethical concerns related to embryonic stem cell use and has the major advantage that...Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors...Mouse Direct Reprogramming of Fibroblasts into Embryonic Sertoli-like Cells by Defined Factors. Cell Stem...via microRNA-dependent and -independent pathways. EMBO Mol Med. 2017 Aug;9(8):1117-1131. Parmar Fibroblasts...

...Transfer proteins from the gel to a membrane. Incubate the membrane with an unconjugated primary antibody...block the membrane to remove excess antibody and prevent unwanted binding. Incubate the membrane with a ...Wash and block the membrane. Activate the HRP with a substrate kit. Image the membrane using either X-ray...tears, saliva, mucus) where it protects mucosal membranes Dimer with a valency of 4 IgA1; IgA2 IgD Coexpressed...short peptide linkers. Diabodies can be used for assembling protein nanostructures. Figure 2: An antibody...recognizes your primary antibody source species. Remember that recombinant antibodies will also have a source...

...regulators of membrane traffic, they also play critical roles at cilia, centrosomes, the plasma membrane, lipid... collection of ARF family plasmids. ARF family members include regulatory GTPases, GEFs, and GAPs. Plasmid...regulatory GTPases have long been characterized as members of the RAS superfamily, though recently have been...signaling activity. Locations of ARF Superfamily members. Figure from Szutl, et al 2019 PMID: 31084567 .... . More information about ARF Superfamily members can be found in: ARF GTPases and their GEFs and GAPs...

...e.g. maize and Arabidopsis) and vectors for easy assembly of one or more gRNA expression cassettes - Qi-Jun...intronized Cas9 series of vectors and DNA modules for assembly of CRISPR/Cas9 constructs - Sylvestre Marillonnet...fluorescent seed selection and multiplex CRISPR/Cas9 assembly for fast generation of multiple Arabidopsis mutants...creating plant expression vectors: A modular plasmid assembly kit for multigene expression, gene silencing, ...Parts II and Infrastructure Kit for hierarchical assembly of plant multigene constructs - Johannes Stuttmann...cloning chassis and set of functional parts for assembling plant transformation vectors - Daniel Voytas....

...expression vectors for TAL effector repeat arrays assembled using the REAL, REAL-Fast or FLASH methods...fragments encoding TAL effector repeat arrays (assembled using the REAL, REAL-Fast or FLASH methods) can...Additional details about the REAL, REAL-Fast, and FLASH assembly methods can be found on the TALengineering website...amino acid sequences (i.e. only the details of the assembly are different among the three methods, the final...REAL TALE Repeat Plasmids in the Joung Lab REAL Assembly TALEN Kit . Vectors encoding wild-type FokI nuclease...

...Adenoviral A block for the AdenoBuilder genome assembly system. Block 6 with deletion in E3 and insertion...Adenoviral A block for the AdenoBuilder genome assembly system; consolidates the regions present in pAd5...Adenoviral A block for the AdenoBuilder genome assembly system. Block 1 with deletion in E1 and insertion...adenovirus serotype 5 (HAdV5) genome that can be assembled in a one-tube reaction. ID Kit Description PI ... Plasmids contain modular inserts for seamless assembly of multiple mutations, alterations, or transgene...

...deposited tools and resources, or read our guide on assembly standards. Browse Plasmids by Microbe System Plasmids...chromosomes - Endy and Shearwin Labs Ralstonia-GG-Kit : Assembly of Ralstonia solanacearum species complex (Rssc...lipolytica Golden Gate tool kit : Integrative vector assembly of up to three transcription units in the oleaginous...circuit design in E. coli - Freemont Lab MoClo-YTK : Assembly of single and multi-gene constructs for S. cerevisiae...perform your synbio experiment. Synthetic Biology Assembly Standards Guide Bacterial Expression Systems :...

...Methods, September 2007, Vol. 4 No. 9, pp. 741-6 Shemiakina et al. : Nature Communications, November 2012,...Addgene has assembled a collection of empty plasmid backbones with different fluorescent tags for you...result in a diverse set of colors. Addgene has assembled a collection of empty plasmid backbones with different...Stepanenko et al. : BioTechniques, Vol. 51, No. 5, November 2011, pp. 313–327 Scholarpedia : Entry by Robert...Shaner et al. : Journal of Cell Science, Vol. 120, December 2007, pp. 4247-4260 Shaner et al. : Nature Methods...

...method Modular Assembly Engineering Reagents - Engineer zinc finger arrays via Modular Assembly method Nuclease...finger proteins deposited by Zinc Finger Consortium members like the Joung Lab, Voytas Lab, and Wolfe Lab Plasmid... engineered zinc finger technology. Consortium members Keith Joung and Daniel Voytas have deposited at...

...human genome with approximately 450 identified members. Many SLCs have mutations associated with disease... window) is a public-private partnership of 13 members from industry and academia financed by the Innovative...generate antibody/ high-affinity ligands. Data is embedded in an integrated knowledgebase that features, ...Superti-Furga CRISPR knockout library targeting human transmembrane transporters. Feedback Please send your feedback...

...community. MCC members gain access to mentors, virtual and local professional development, membership to a local...research opportunities to their students and other members of the MCC community. They support educators interested... CUREs, and opportunities to mentor future MCC members. Find a regional hub (Link opens in a new window...