Synthetic Biology TOOLS TO SUPPORT DESIGN AND ASSEMBLY

Transcription

1 Synthetic iology TOOLS TO SUPPORT DESIGN ND SSEMLY

2 0 0 0 SYNTHETIC IOLOGY Synthetic iology Tools to support design and assembly The goal of synthetic biology, in which genes and proteins are viewed as parts or devices, is redesigning and/or assembling them in novel ways to create a new and useful functionality. Recent advances in the production of biochemicals and biofuels, and a new understanding of the minimal genome, benefit from synthetic biological approaches. These projects often rely on the ordered assembly of multiple DN sequences to create large, artificial DN structures, and methods have evolved to simplify this process. New England iolabs now offers several products that can be used for DN assembly and cloning. Use this chart to determine which product would work best to assemble your DN. NEuilder HiFi DN ssembly Gibson ssembly NE Golden Gate ssembly Mix USER Enzyme NE #E262 NE #E5520 NE #E550 NE #E26 NE #E600 NE #M5505 PROPERTIES Removes or End Mismatches *** * N/ N/ ssembles with High Fidelity at Junctions *** ** *** *** Tolerates Repetitive Sequences at Ends * * *** *** Generates Fully Ligated Product *** *** *** NR Joins dsdn with Single-stranded Oligo *** ** NR NR ssembles with High Efficiency with Low mounts of DN *** ** ** ** ccommodates Flexible Overlap Lengths *** *** * ** PPLICTIONS Simple Cloning (-2 Fragments) *** *** *** *** 4-6 Fragment ssembly *** *** *** *** >6 Fragment ssembly *** ** *** *** Template Construction for In vitro Transcription *** *** *** * Synthetic Whole Genome ssembly *** * * * Multiple Site-directed Mutagenesis *** ** ** ** Library Generation ** ** ** ** Pathway Engineering *** ** ** *** TLENs ** ** *** ** Short Hairpin RN Cloning (shrn) *** ** * * grn Library Generation *** ** * * Large Fragment (>0 kb) ssembly *** *** *** ** Small Fragment (<00 bp) ssembly *** * *** *** Use in Successive Rounds of Restriction Enzyme ssembly *** * NR * KEY *** Optimal, recommended product for selected application N/ Not applicable to this application ** Works well for selected application NR Not recommended Will perform selected application, but is not recommended * NE offers several online tools to aid in DN assembly. Look for the icons on the following pages. 2

3 SYNTHETIC IOLOGY NEuilder HiFi DN ssembly NEuilder HiFi DN ssembly enables virtually error-free joining of DN fragments, even those with - and -end mismatches. vailable with and without competent E. coli, this flexible kit enables simple and fast seamless cloning utilizing a new proprietary high-fidelity polymerase. Make NEuilder HiFi your first choice for DN assembly and cloning. Overview of the NEuilder HiFi DN ssembly cloning method NEuilder HiFi DN ssembly Cloning Kit (NE #E5520) NEuilder HiFi DN ssembly Master Mix (NE #E262) Simple and fast seamless cloning in as little as 5 minutes DN Preparation From: PCR Restriction enzyme digestion Synthetic DN (e.g., glocks) NEuilder HiFi DN ssembly Master Mix Single-tube reaction Exonuclease chews back ends to create single-stranded overhangs DN polymerase fills in gaps within each annealed fragment DN ligase seals nicks in the assembled DN Transformation Linear vector + Incubate at 50 C for 5-60 minutes DN inserts with 5-30 bp overlapping ends (PCR-amplified) ssembled DN C C Use one system for both "standard-size" cloning and larger gene assembly products (up to 2 fragments and 20 kb) DN can be used immediately for transformation or as template for PCR or RC dapts easily for multiple DN manipulations, including site-directed mutagenesis Enjoy less screening/re-sequencing of constructs, with virtually error-free, high-fidelity assembly Use NEuilder HiFi in successive rounds of assembly, as it removes - and -end mismatches ridge two ds-fragments with a synthetic ssdn oligo for simple and fast construction (e.g., linker insertion or grn library) No licensing fee requirements from NE for NEuilder products DN nalysis OR OR NEuilder HiFi DN ssembly Cloning Kit includes the NEuilder HiFi DN ssembly Master Mix and NE 5-alpha Competent E. coli RE Digest Colony PCR Sequencing TOOLS & RESOURCES Visit NEuilderHiFi.com to find: NEuilder HiFi DN ssembly offers improved efficiency and accuracy with lower amounts of DN by increasing overlap length NEuilder HiFi DN ssembly Master Mix Gibson ssembly Master Mix Online tutorials to help with assembly and primer design pplication notes utilizing NEuilder HiFi ccess to NEuilder ssembly Tool, our online primer design tool Number of Colonies Length in overlap (bp) mount used (fmol) Reactions were set up in a 4-fragment assembly reaction according to recommended reaction conditions. mount of DN and size of overlap is shown INTRODUCTION TO NEUILDER HIFI DN SSEMLY For help with designing primers, try NEuilder ssembly Tool at NEuilder.neb.com 3

4 0 0 0 SYNTHETIC IOLOGY Gibson ssembly Gibson ssembly enables multiple, overlapping DN fragments to be joined in a single-tube isothermal reaction, with no additional sequence added (scar-less). The Gibson ssembly Master Mix includes three different enzymatic activities that perform in a single buffer (described below). The assembled, fully-sealed construct is then transformed into NE 5-alpha competent E. coli. The entire protocol, from assembly to transformation, takes just under two hours. Gibson ssembly cloning method Gibson ssembly Cloning Kit (NE #E550) Gibson ssembly Master Mix (NE #E26) ssemble multiple fragments and transform in just under two hours Clone into any vector with no additional sequence added dsdn fragments with overlapping ends Gibson ssembly Master Mix Incubate at 50 C for 5-60 minutes Gibson ssembly Exonuclease chews back ends. DN fragments anneal. DN polymerase extends ends. Gibson ssembly Cloning Kit includes the Gibson ssembly Master Mix and NE 5-alpha Competent E. coli No PCR cleanup step required TOOLS & RESOURCES Visit NEGibson.com to find: Online tutorials to help with assembly and primer design pplication notes utilizing Gibson ssembly ccess to NEuilder ssembly Tool, our online primer design tool DN ligase seals nicks. Fully ssembled DN Gibson ssembly Cloning Kit provides robust transformation efficiencies INTRODUCTION TO GISON SSEMLY Number of recombinant colonies (x0 3 ) Gibson ssembly In-Fusion Genert For help with designing primers, try NEuilder ssembly Tool at NEuilder.neb.com 0 0 kb 3kb 5 kb Fragment size ssembly reactions containing 25 ng of linear puc9 vector and 0.04 pmol of each fragment were performed following individual suppliers recommended protocols and using the competent cells provided with the kit. The total number of recombinant colonies was calculated per 25 ng of linear puc9 vector added to the assembly reaction. Some components of this product are manufactured by New England iolabs, Inc. under license from Synthetic Genomics, Inc. 4

5 SYNTHETIC IOLOGY NE Golden Gate ssembly The efficient and seamless assembly of DN fragments, commonly referred to as Golden Gate assembly (,2), has its origins in 996 when, for the first time, it was shown that multiple inserts could be assembled into a vector backbone using only the sequential (3) or simultaneous (4) activities of a single Type IIS restriction enzyme and T4 DN Ligase. This method can be accomplished using Type IIS restriction enzymes, such as sai, and can also be used for the cloning of single inserts. The method is efficient and can be completed in one tube in as little as 5 minutes for single inserts, or can utilize cycling steps for multiple inserts. The NE Golden Gate ssembly Mix incorporates digestion with sai and ligation with T4 DN Ligase into a single reaction, and can be used to assemble up to 0 fragments in a single step. NE Golden Gate ssembly workflow NE Golden Gate ssembly Mix (NE #E600) Seamless cloning no scar remains following assembly Ordered assembly of up to 0 fragments in a single reaction Efficient with regions with high GC content and areas of repeats Compatible with a broad range of fragment sizes (< 00 bp to > 5 kb) TOOLS & RESOURCES sai sai Visit to find: NNNNNGGCC NNNNNCTCTGG P GGTCTCNNNNN CCGGNNNNN P2 P3 P4 P5 P6 Publications and protocols related to Golden Gate ssembly ccess to NE Golden Gate ssembly Tool, our online assembly tool PCR amplification of vector and fragments Single-tube reaction sai DN ligase PCRlinearized vector + PCR-amplified fragments In its simplest form, Golden Gate ssembly requires a Type IIS recognition site, in this case, sai (GGTCTC), added to both ends of a dsdn fragment. fter digestion, these sites are left behind, with each fragment bearing the designed 4-base overhangs that direct the assembly. NE Golden Gate ssembly Mix offers improved assembly INTRODUCTION TO GOLDEN GTE SSEMLY Speed up your experimental design with our online assembly tool at NEGoldenGate.neb.com 7,000 6,000 6,560 5,750 NE Golden Gate ssembly Mix (NE #E600) Invitrogen Genert Type IIs ssembly Kit, sai Number of Transformants per Plate 5,000 4,000 3,000 2,000, ,370 3, Precloned ( hour, 37 C) Precloned (30 cycles/ min. steps),663,636 ssembly Protocol 3 2 mplicons ( hour, 37 C), mplicons (30 cycles/ min. steps) ssembly reactions were set up using either precloned inserts or PCR amplicons directly. Reaction conditions were set up according to manufacturer, and are shown above. Two separate experiments are shown for each reaction type. References:. Engler, C. et al. (2008) PLoS ONE, 3: e Engler, C. et al. (2009) PLoS ONE, 4: e Lee, J.H. et al. (996) Genetic nalysis: iomolecular Engineering, 3; Padgett, K.. and Sorge, J.. (996) Gene, 68,

6 0 0 0 SYNTHETIC IOLOGY USER Enzyme The USER-friendly DN engineering method enables multiple PCR fragment assembly, nucleotide sequence alteration and directional cloning. Target DN molecules and cloning vector are generated by PCR with 6-0 bases of homology between the neighboring fragments. PCR primers contain a single deoxyuracil residue (du) flanking the end of the homology region, and can accommodate nucleotide substitutions, insertions and/or deletions. The primers are then used to amplify the vector and target DN with discrete overlapping fragments that incorporate a du at each end. Subsequent treatment of PCR fragments with USER Enzyme creates a single nucleotide gap at each du, resulting in PCR fragments flanked with ss-extensions that allow seamless and directional assembly of customized DN molecules into a linearized vector. Multi-fragment assemblies and/or various mutagenic changes can be performed in a single experiment. USER Enzyme (NE #M5505) Seamless and directional assembly Multiple fragment assemblies and/or mutations can be performed in a single experiment USER assembly is performed at 37 C or room temperature (no need for thermocycler) USER method can be used for assembly of small fragments (< 00bp) or oligo duplexes and for sequences with end repeats. DN assembly with USER Enzyme n optional TG should be included TG if protein expression is desired. Left primer GGGCUNNNNNN Target DN NNNNNNUGGGG Right primer PCR GGGCU CCTCTGT CTTTCCC UGGGG USER CCTCTGT CTTTCCC ssembly pne206 ssembled DN Transformation iorick ssembly With the iorick synthetic biology approach, DN fragments encoding proteins, promoters, ribosome binding sites, etc., have been standardized and are contained in a "parts" registry of plasmids with identical restriction sites flanking the "payload" of the part. y employing standardized flanking sites that are not contained within the coding sequence of the part, they can be ligated in any order to create a novel "device". y choosing restriction sites with compatible ends that destroy the recognition site when ligated to one another, parts can be combined together and the original flanking sites re-used for the next round of assembly. Despite the limitations of introducing a sequence scar for every ligation event and the multiple rounds of assembly required to fabricate a device, a wide assortment of exciting systems have been designed and built. iorick ssembly Kit (NE #E0546) The iorick ssembly Kit was developed in partnership with Ginkgo ioworks The iorick approach has been used to build a wide variety of novel biological systems For more details and for technical questions, please visit ginkgobioworks.com/support 6

7 SYNTHETIC IOLOGY Cas9 Nuclease, S. pyogenes Cas9 Nuclease is a double stranded DN nuclease and central component of CRISPR(clustered regularly interspaced short palindromic repeat)-based immunity. In bacteria and archea, the CRISPR/Cas9 system functions to protect cells from foreign DN. CRISPR genomic loci are transcribed and processed into guide RNs that are incorporated into Cas9 Nuclease. Guide RNs direct the Cas9 nuclease to its target by complementary base pairing. Cas9 Nuclease has been adapted for use in genome engineering, because it can be easily programmed for target specificity by supplying guide RNs of almost any sequence. In vitro, Cas9 permits flexible, user-specified, introduction of double stranded breaks at ~20 bp recognition sequences. In cells and animals, genome editing is performed by expressing Cas9 Nuclease and guide RN (often singleguide RN sgrn) from DN constructs (plasmid or virus), supplying RN encoding Cas9 nuclease and sgrn, or by introducing RN-programmed Cas9 Nuclease directly. Targeted doubled stranded breaks can be repaired by nonhomologous end joining (NHEJ), knocking out gene function and enabling large scale deletions, or homology-directed repair (HDR) in the presence of an HDR template, enabling targeted insertions or substitutions. Cas9 Nuclease, S. pyogenes sequence recognition and DN cleavage Cas9 Nuclease, S. pyogenes (NE #M0386) vailable in standard (NE #M0386S/L) and high concentration (NE #M0386M) Coming soon: Cas9 with nuclear localization signal (NLS) TOOLS & RESOURCES Visit to find: Up-to-date listing of products and protocols to support this application Tips for planning your Cas9 experiment Strategies for sgrn template construction for Cas9 gene editing Protocols for measuring targeting efficiency with the T7 Endonuclease I ssay Cas9 Nuclease, S. pyogenes DN PM (NGG) CGCTTGTTTCGGCGTGG GTTGG Cleavage Cleavage GCGCGCCGCCC CTCC CGCUUGUUUCGGCGUGG GU Nucleotide position (upstream of PM) sgrn PURExpress In Vitro Protein Synthesis Kit rapid method for gene expression analysis, PURExpress is a novel cell-free transcription/translation system reconstituted from purified components necessary for E. coli translation. Express a wide range of proteins free of modification or degradation by simply mixing two tubes followed by the addition of template DN. With results available in only a few hours, PURExpress saves valuable laboratory time and is ideal for high throughput technologies. Product selection includes the original kit, with all components in two tubes, as well as options for protein translation experiments, protein synthesis/ribosomal display experiments and synthesis with modified amino acids. PURExpress In Vitro Protein Synthesis Kit (NE #E6800) Suitable for circular or linear DN template Visualize synthesized protein directly on a Coomassie stained gel Protein expression in approximately 2 hours Transcription/translation components can be removed by affinity chromatography 7

8 Ordering Information Ordering Information PRODUCT NE # SIZE NEuilder HiFi DN ssembly Cloning Kit E5520S 0 reactions NEuilder HiFi DN ssembly Master Mix E262S/L/X 0/50/250 reactions Gibson ssembly Cloning Kit E550S 0 reactions Gibson ssembly Master Mix E26S/L 0/50 reactions NE Golden Gate ssembly Mix E600S 5 reactions RECOMMENDED RESOURCES Programming Life: Inquiry & Engineering Through Synthetic iology Follow the evolution of synthetic biology by visiting our feature article at USER Enzyme M5505S/L 50/250 units iorick ssembly Kit E0546S 50 reactions Cas 9 Nuclease, S. pyogenes M0386S/L/M 50/250/500 pmol PURExpress In Vitro Protein Synthesis Kit E6800S/L 0/00 reactions PURExpress Ribosome Kit E333S 0 reactions PURExpress (aa, trn) Kit E6840S 0 reactions PURExpress RF23 Kit E6850S 0 reactions PURExpress Disulfide ond Enhancer E6820S 50 reactions E. coli Ribosome P0763S mg dditional Reagents to Facilitate Synthetic iology Experiments PRODUCT NE # SIZE RESTRICTION ENZYMES sai R0535S/L,000/5,000 units sai-hf R3535S/L,000/5,000 units bsi R0539S/L 300/,500 units Training the Next Generation of ioengineers Since its inception in 2004, igem has evolved into a highly successful vehicle for training and showcasing a new generation of biological engineers using the synthetic biology framework To date, more than 28,000 students have participated in this competition Teams are supported by various organizations, including NE For more information, visit smi R0580S/L 200/,000 units DN LIGSES & MODIFYING ENZYMES Thermos aquaticus (Taq) DN Ligase M0208S/L 2,000/0,000 units T4 DN Ligase M0202S/L/T/M 20,000/00,000 units T5 Exonuclease M0363S/L,000/5,000 units COMPETENT CELLS NE 5-alpha Competent E. coli (High Efficiency) C2987H/I/P 20 x 0.05 ml/6 x 0.2 ml/ x 96 well plate NE 0-beta Competent E. coli (High Efficiency) C309H/I 20 x 0.05 ml/6 x 0.2 ml NE Stable Competent E. coli (High Efficiency) C3040H/I 20 x 0.05 ml/6 x 0.2 ml DN LDDERS Quick-Load Purple 2-log DN Ladder ( kb) N0550S gel lanes Quick-Load Purple 00 bp DN Ladder N055S 25 gel lanes Quick-Load Purple kb DN Ladder N0552S 25 gel lanes NEW ENGLND IOLS, NE, HF, NEUILDER, PUREXPRESS and QUICK-LOD are registered trademarks of New England iolabs, Inc. GENERT is a registered trademark of Life Technologies, Inc. GISON SSEMLY is a registered trademark of Synthetic Genomics, Inc. IN-FUSION is a registered trademark of Clontech Laboratories, Inc. IORICK is a registered trademark of The ioricks Foundation ( USER is a trademark of New England iolabs, Inc. GINKGO IOWORKS is a trademark of Ginkgo ioworks, Inc. ISO 900 Registered Quality Management ISO 400 Registered Environmental Management ISO 3485 Registered Medical Devices New England iolabs, Inc., 240 County Road, Ipswich, M Telephone: (978) Toll Free: (US Orders) Toll Free: (US Tech) Fax: (978) info@neb.com SYN_IO Version.0 5/5