BBF RFC 53: USTC MetaPart Assembly Standard -- Extending RFC 10 to Enable Scarless Protein Fusion with Type IIS Restriction Enzyme EarI and SapI

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1 BBF RFC 53: -- Extending RFC 10 to Enable Scarless Protein Fusion with Type IIS Restriction Enzyme and Hao Jiang, Yang Zhang, Ruijun Zhu, Chang Liu, Duo An, Ying Zhang, Ge Gao, and Jing Yang 5 November Purpose The original BioBrick assembly standard in BBF RFC 10 has provided a great solution to the construction of long DNA sequences by combination of basic DNA parts. Thousands of parts have been constructed following this standard. However, BBF RFC 10 has some shortcomings, the most important one is the incompatibility with protein fusion. To overcome these shortcomings, a lot of new standards have been developed, such as BBF RFC 12, 15, 20, 21, 22, 23, 25, 26, 28, 37, 38, 39, 44, 45, 46, 47, 54, 61 and 62. Unfortunately, these standards also have deficiencies, none of these standards has enough advantages to become a new de facto standard. A better standard successor is still in great need.[1] In BBF RFC 53, a new assembly standard with the following features is described: Compatible with BBF RFC 10 (Assembly Standard 53 parts are Assembly Standard 10 parts, many Assembly Standard 10 parts are Assembly Standard 53 parts). Support protein fusion. Assembly scar is at most 3bp (1 amino acid scar) and can be in the native sequence (scarless), because cohesive ends can be any sequences for type IIS enzymes. N-terminals are native coding sequences. Support scarless assembly between eukaryotic RBS Kozak sequences and protein coding sequences. Support fusion of elements for promoters, riboregulators, and other special parts. Support standard measurement and screening or selection systems. (See 5.2.2) 1

2 Enzymes used are not too expensive. All ligations are cohesive end ligations. Diverse and flexible assembly strategies. Less complicated than BioBricks++ assembly standard.[2] Can be used as a de novo DNA synthesis method and a site-directed mutagenesis method, because of the scarless feature, so no other de novo DNA synthesis or site-directed mutagenesis methods is needed in part construction. (See 6.7 and 6.8) 2. Relation to other BBF RFCs BBF RFC 53 updates BBF RFC Copyright Notice Copyright (C) The BioBricks Foundation (2010). All Rights Reserved. 4. Enzymes Besides the 5 restriction enzymes used in Assembly Standard 10, 4 more enzymes are used: SacI, BglII,, and. The cutting sites of the 9 enzymes are listed below: EcoRI XbaI SpeI PstI NotI 5'-GAATTC-3' 5'-TCTAGA-3' 5'-ACTAGT-3' 5'-CTGCAG-3' 5'-GCGGCCGC-3' 3'-CTTAAG-5' 3'-AGATCT-5' 3'-TGATCA-5' 3'-GACGTC-5' 3'-CGCCGGCG-5' SacI BglII 5'-GAGCTC-3' 5'-AGATCT-3' 5'-CTCTTCNNNN-3' 5'-GCTCTTCNNNN-3' 3'-CTCGAG-5' 3'-TCTAGA-5' 3'-GAGAAGNNNN-5' 3'-CGAGAAGNNNN-5' and are type IIS restriction enzymes, their recognition sequences are asymmetric, and the cleavage sites are out of the recognition sequences to one side. This makes type IIS restriction enzymes very useful, many cloning methods based on them have been developed.[3-7] The recognition sequence contains the recognition sequence, so can cut at sites, but cannot cut sites. SacI and BglII recognition sequences contain overlap sequences with and, so they can be used to inactivate and sites. (See below for details.) Fermentas FastDigest enzymes are RECOMMENDED. They are more convenient and less expensive than NEB enzymes. 2

3 5. Parts 5.1. Requirements An Assembly Standard 53 part (or Assembly Standard 53 plasmid backbone) MUST meet all requirements for an Assembly Standard 10 part (or Assembly Standard 10 plasmid backbone) The sequence of an Assembly Standard 53 part MUST meet one of the 10 formats in Except the defined sequences in 5.2, an Assembly Standard 53 part (or Assembly Standard 53 plasmid backbone) MUST NOT include the following sequences: 5 -GAATTC-3 (EcoRI site) 5 -TCTAGA-3 (XbaI site) 5 -ACTAGT-3 (SpeI site) 5 -CTGCAG-3 (PstI site) 5 -GCGGCCGC-3 (NotI site) 5 -CTCTTC-3 ( site) 5 -GAAGAG-3 ( site) 5 -GAGCTC-3 (SacI site) 5 -AGATCT-3 (BglII site) The following sequences MAY also be eliminated if convenient: 5 -GCTAGC-3 (NheI site) 5 -CCTAGG-3 (AvrII site) 5 -GAGGAG-3 (BseRI site) 5 -CTCCTC-3 (BseRI site) 5 -CTTGAG-3 (BpuEI site) 5 -CTCAAG-3 (BpuEI site) 5 -GGCGCGCC-3 (AscI site) 5 -CATATG-3 (NdeI site) 5 -GGTCTC-3 (Eco31I site) 5 -GAGACC-3 (Eco31I site) 5 -GAAGAC-3 (BbsI site) 5 -GTCTTC-3 (BbsI site) 3

4 5.2. Formats There are 4 basic formats (Meta, Head, Tail, and Full) and 6 composite formats (XM, MX, XMX, HX, XT, and HXT). The following terms are used to define the formats: Prefix22(PF22): 5 -GAATTCGCGGCCGCTTCTAGAG-3, a 22bp sequence before a Part. Prefix20(PF20): 5 -GAATTCGCGGCCGCTTCTAG-3, a 20bp sequence before a Part, MUST be followed by 5 -ATG-3. Prefix22/20(PF22/20): 5 -GAATTCGCGGCCGCTTCTAGAK-3, it can be either PF22(5 -GAATTCGCGGCCGCTTCTAGAG-3 ) or PF20+AT(5 - GAATTCGCGGCCGCTTCTAGAT-3 ). Suffix(SF): 5 -TACTAGTAGCGGCCGCTGCAG-3, a 21bp sequence after a Part. MetaPrefix(MPF): 5 -GAGCTCTTCA-3, a 10bp sequence before a MetaPart or a TailPart. MetaSuffix(MSF): 5 -TGAAGAGATC-3, a 10bp sequence after a MetaPart or a HeadPart. Part: the sequence after a PF22 or PF20, and before a SF. MetaPart(MP): the sequence after an MPF, and before an MSF. HeadPart(HP): the sequence after a PF22 or PF20, before an MSF, and without sequences in (thus without MPF). TailPart(TP): the sequence after an MPF, before a SF, and without sequences in (thus without MSF). FullPart(FP): the sequence after a PF22 or PF20, before a SF, and without sequences in (thus without MPF and MSF). ExtraPrefix(XPF): the sequence ends with MPF, and after a PF22 or PF20. ExtraSuffix(XSF): the sequence starts with MSF, and before a SF. ExtraInfix(XIF): the sequence starts with MSF, and ends with MPF. : the first 3bp of MetaPart or TailPart. : the last 3bp of MetaPart or HeadPart. S3: the 3bp scar from the fusion of an and an. S8: 5 -TACTAGAG-3, the 8bp scar from BBF RFC 10 assembly. S6: 5 -TACTAG-3, the 6bp scar from BBF RFC 10 assembly, MUST be followed by 5 -ATG-3. 4

5 S8/6: 5 -TACTAGAK-3, it can be either S8(5 -TACTAGAG-3 ) or S6+AT(5 - TACTAGAT-3 ) Basic Formats Meta Format Prefix22 Suffix EcoRI NotI XbaI SpeI NotI PstI 5'...GAATTCGCGGCCGCTTCTAGAGGAGCTCTTCANNN...NNNTGAAGAGATCTACTAGTAGCGGCCGCTGCAG...3' 3'...CTTAAGCGCCGGCGAAGATCTCCTCGAGAAGTNNN...NNNACTTCTCTAGATGATCATCGCCGGCGACGTC...5' Part MetaPart Meta format is the most important format and the RECOMMENDED format. MetaPart in this format has the potential of fusion both before it and after it. Examples: BBa_K ~ BBa_K371017, BBa_K ~ BBa_K371032, BBa_K371035, BBa_K371036, BBa_K371043, BBa_K371044, BBa_K371053, BBa_K371054, BBa_K371061, BBa_K Head Format Part HeadPart Prefix22/20 MetaSuffix Suffix EcoRI NotI XbaI BglII SpeI NotI PstI 5'...GAATTCGCGGCCGCTTCTAGAK...NNNTGAAGAGATCTACTAGTAGCGGCCGCTGCAG...3' 3'...CTTAAGCGCCGGCGAAGATCTM...NNNACTTCTCTAGATGATCATCGCCGGCGACGTC...5' HeadPart in this format has the potential of fusion after it. Examples: BBa_K371057, BBa_K Tail Format Part TailPart Prefix22 MetaPrefix Suffix EcoRI NotI XbaI SpeI NotI PstI 5'...GAATTCGCGGCCGCTTCTAGAGGAGCTCTTCANNN...TACTAGTAGCGGCCGCTGCAG...3' 3'...CTTAAGCGCCGGCGAAGATCTCCTCGAGAAGTNNN...ATGATCATCGCCGGCGACGTC...5' 5

6 TailPart in this format has the potential of fusion before it. Examples: BBa_K Full Format Prefix22/20 Part FullPart Suffix EcoRI NotI XbaI SpeI NotI PstI 5'...GAATTCGCGGCCGCTTCTAGAK...TACTAGTAGCGGCCGCTGCAG...3' 3'...CTTAAGCGCCGGCGAAGATCTM...ATGATCATCGCCGGCGACGTC...5' If a BBF RFC 10 part doesn t have sequences in 5.1.3, it is a BBF RFC 53 FullPart. FullPart doesn t have the potential of fusion. Head format, tail format and full format are used to comply with old parts. Meta format is RECOMMENDED for all new parts (not only for coding parts) Composite Formats Composite format parts are products of BBF RFC 10 assembly with basic format parts. These formats are useful, because extra parts or devices can be added beside the part in these formats, while don t keep the part from further fusion or assembly, which is not possible in BBF RFC 10. Measurement or screening/selection devices [8] can be built using these formats. XPF and XSF can be used to send PoPS input (e.g. a promoter) and receive PoPS output (e.g. reporter), while XIF can be used to receive PoPS output and send new PoPS inputs (e.g. inverter). XIF can also carry screening or selection markers such as BBa_P1010 (ccdb generator) and BBa_J04450 (mrfp generator). The MPF and MSF are designed to contain an RBS and a stop codon. This makes it possible to test protein expression before assembly. Can Be Start Codon MetaPart RBS Stop Codon 5'...CTAGAGGAGCTCTTCANNN...NNNTGAAGAGATCTACTAG...3' 3'...GATCTCCTCGAGAAGTNNN...NNNACTTCTCTAGATGAAG...5' XM Format 6

7 ExtraPrefix Prefix22/20 S8 Suffix EcoRI NotI XbaI SpeI NotI PstI 5'...GAATTCGCGGCCGCTTCTAGAK...TACTAGAGGAGCTCTTCANNN...NNNTGAAGAGATCTACTAGTAGCGGCCGCTGCAG...3' 3'...CTTAAGCGCCGGCGAAGATCTM...ATGATCTCCTCGAGAAGTNNN...NNNACTTCTCTAGATGATCATCGCCGGCGACGTC...5' Part MetaPart Assembly to produce XM: Full + Meta = XM XT + Head = XM Full + XM =XM MX Format Part MetaPart ExtraSuffix Prefix22 S8/6 Suffix EcoRI NotI XbaI SpeI NotI PstI 5'...GAATTCGCGGCCGCTTCTAGAGGAGCTCTTCANNN...NNNTGAAGAGATCTACTAGAK...TACTAGTAGCGGCCGCTGCAG...3' 3'...CTTAAGCGCCGGCGAAGATCTCCTCGAGAAGTNNN...NNNACTTCTCTAGATGATCTM...ATGATCATCGCCGGCGACGTC...5' Assembly to produce MX: Meta + Full = MX Tail + HX = MX MX + Full = MX XMX Format Part ExtraPrefix MetaPart ExtraSuffix Prefix22/20 S8 S8/6 Suffix EcoRI NotI XbaI SpeI NotI PstI 5'...GAATTCGCGGCCGCTTCTAGAK...TACTAGAGGAGCTCTTCANNN...NNNTGAAGAGATCTACTAGAK...TACTAGTAGCGGCCGCTGCAG...3' 3'...CTTAAGCGCCGGCGAAGATCTM...ATGATCTCCTCGAGAAGTNNN...NNNACTTCTCTAGATGATCTM...ATGATCATCGCCGGCGACGTC...5' Assembly to produce XMX: XM + Full = XMX Full + MX = XMX XT + HX = XMX Full + XMX = XMX XMX + Full = XMX 7

8 HX Format HeadPart Part ExtraSuffix Prefix22/20 MetaSuffix S8/6 Suffix EcoRI NotI XbaI BglII SpeI NotI PstI 5'...GAATTCGCGGCCGCTTCTAGAK...NNNTGAAGAGATCTACTAGAK...TACTAGTAGCGGCCGCTGCAG...3' 3'...CTTAAGCGCCGGCGAAGATCTM...NNNACTTCTCTAGATGATCTM...ATGATCATCGCCGGCGACGTC...5' Assembly to produce HX: Head + Full = HX HX + Full = HX Full + HX = HX XT Format Part ExtraPrefix Prefix22/20 S8 MetaPrefix Suffix EcoRI NotI XbaI SpeI NotI PstI 5'...GAATTCGCGGCCGCTTCTAGAK...TACTAGAGGAGCTCTTCANNN...TACTAGTAGCGGCCGCTGCAG...3' 3'...CTTAAGCGCCGGCGAAGATCTM...ATGATCTCCTCGAGAAGTNNN...ATGATCATCGCCGGCGACGTC...5' TailPart Assembly to produce XT: Full + Tail =XT XT + Full = XT Full +XT = XT HXT Format Part HeadPart ExtraInfix TailPart Prefix22/20 MetaSuffix S8 MetaPrefix Suffix EcoRI NotI XbaI BglII SpeI NotI PstI 5'...GAATTCGCGGCCGCTTCTAGAK...NNNTGAAGAGATCTACTAGAGGAGCTCTTCANNN...TACTAGTAGCGGCCGCTGCAG...3' 3'...CTTAAGCGCCGGCGAAGATCTM...NNNACTTCTCTAGATGATCTCCTCGAGAAGTNNN...ATGATCATCGCCGGCGACGTC...5' or 8

9 Part HeadPart ExtraInfix TailPart Prefix22/20 MetaSuffix S8/6 S8 MetaPrefix Suffix EcoRI NotI XbaI BglII SpeI NotI PstI 5'...GAATTCGCGGCCGCTTCTAGAK...NNNTGAAGAGATCTACTAGAK...TACTAGAGGAGCTCTTCANNN...TACTAGTAGCGGCCGCTGCAG...3' 3'...CTTAAGCGCCGGCGAAGATCTM...NNNACTTCTCTAGATGATCTM...ATGATCTCCTCGAGAAGTNNN...ATGATCATCGCCGGCGACGTC...5' Assembly to produce HXT: Head + Tail = HXT HX + Tail = HXT Head + XT = HXT HX + XT = HXT Full + HXT = HXT HXT + Full = HXT Example: BBa_K and BBa_K are not standard HXT parts (and even not Assembly Standard 53 parts), but they are similar with HXT format, and can be used similarly. (See ) 5.3. D-L standard parts To make a fusion of two parts, the of the first part and the of the second part MUST be the same. Therefore, it is better to use the same set of triplets as and, so that parts can be fused with each other. Two special triplets are carefully chosen: ATG (start codon and coding for methionine) and GGT (coding for glycine). According to the and, parts using these 2 triplets can be divided into 4 types: D, L, DL and LD. D is for Domain-Like, L is for Linker-Like, DL and LD are similar to the fusion of D and L. Fusion between D and L, D and LD, L and D, L and DL, LD and L, LD and LD, DL and D, DL and DL are possible. Format: D-Meta D-Head D-Tail D-XM D-MX D-XMX D-HX D-XT D-HXT : ATG - ATG ATG ATG ATG - ATG ATG : GGT GGT - GGT GGT GGT GGT - GGT Format: L-Meta L-Head L-Tail L-XM L-MX L-XMX L-HX L-XT L-HXT : GGT - GGT GGT GGT GGT - GGT GGT : ATG ATG - ATG ATG ATG ATG - ATG Format: DL-Meta DL-XM DL-MX DL-XMX DL-HXT : ATG ATG ATG ATG ATG : ATG ATG ATG ATG ATG 9

10 Format: LD-Meta LD-XM LD-MX LD-XMX LD-HXT : GGT GGT GGT GGT GGT : GGT GGT GGT GGT GGT Examples: D-Meta: BBa_K ~ BBa_K371017, BBa_K ~ BBa_K371032, BBa_K371035, BBa_K371036, BBa_K371043, BBa_K371044, BBa_K371053, BBa_K L-Meta: BBa_K371061, BBa_K L-Head: BBa_K371057, BBa_K L-Tail: BBa_K Parts with ATG or GGT as and are called D-L standard parts. Reusable parts are RECOMMENDED to be designed as D-L standard parts, but non-d-l-standard parts MAY also be used. The first step to design a D-L standard part is to choose the type. Protein domain SHOULD be D type. Protein linker SHOULD be L type. The type of a part SHOULD be chosen based on the type of other parts that the part needs to be fused with. The general guide below is RECOMMENDED to be followed: D-Parts: Protein domain (e.g. BBa_K ~ BBa_K371017, BBa_K371043, BBa_K371044) Promoter Device with PoPS output D/L/D fusion product (e.g. BBa_K ~ BBa_K371032, BBa_K371035, BBa_K371036, BBa_K371054) L-Parts: Protein linker (e.g. BBa_K371062) Protein cleavage site Stop codon RBS (e.g. BBa_K371061) Stop codon - Terminator Device with PoPS input L/D/L fusion product DL-Parts: N-terminal protein tag DNA cleavage site Primer 10

11 Special DNA part D/L fusion product LD-Parts: C-terminal protein tag RNA cleavage site Special RNA part Device with PoPS input and PoPS output L/D fusion product The second step to design a D-L standard part is to add the and to the original sequence. For a non-coding part, adding sequence to the end is usually not a problem. For a coding part, ATG is the start codon and usually won t change the native sequence, but for GGT, if the amino acid at the terminal is glycine but the sequence is not GGT, just change the sequence to GGT, if not glycine, add GGT to the end. Adding sequence to the end sometimes will cause problems, if this is the case, there are different choices. One is to use non-d-l-standard parts and keep the native sequence, and special designed linkers can be used for fusions. The other choice is to still use the D-L standard, but add some sequence before add the or, or change the boundary of the part to avoid the special end. For example, include a stop codon for a C-terminal tag, and GGT can be added after the stop codon Plasmid s and Part Construction Currently, psb series plasmid backbones that comply with BBF RFC 53 are only psb1t3, psb3c5, psb3t5 and psb3k5. We have constructed a new version of psb1c3: BBa_K There is only 1bp mutation to eliminate the SacI site and make it comply with BBF RFC 53. The other properties are the same. Using BBa_K instead of psb1c3 is RECOMMENDED. We also constructed a part BBa_K to help the construction of BBF RFC 53 parts in different formats. BBa_K is a D-Meta part with only a NheI site between and. The NheI site can be used to introduce screening or selection markers, such as BBa_J04450 (mrfp generator) and BBa_P1010 (ccdb generator), by digesting with XbaI and SpeI and inserting into the NheI site. BBa_K MetaPart Prefix22/20 MetaPrefix MetaSuffix Suffix EcoRI NotI XbaI NheI SpeI NotI PstI 5'...GAATTCGCGGCCGCTTCTAGAGGAGCTCTTCAATGGCTAGCGGTTGAAGAGATCTACTAGTAGCGGCCGCTGCAG...3' 3'...CTTAAGCGCCGGCGAAGATCTCCTCGAGAAGTTACCGATCGCCAACTTCTCTAGATGATCATCGCCGGCGACGTC...5' 11

12 D-Meta format parts can be constructed by PCR with site in the 5 of primers, digest with, and ligate into corresponding sites in BBa_K Other Meta format parts can be constructed by PCR with MPF and MSF in the 5 of primers, digest with SacI and BglII, and ligate into corresponding sites in BBa_K Head format parts can be constructed by PCR with PF22 or PF20 and MSF in the 5 of primers, digest with XbaI and BglII, and ligate into corresponding sites in BBa_K Tail format parts can be constructed by PCR with MPF and SF in the 5 of primers, digest with SacI and SpeI, and ligate into corresponding sites in BBa_K For parts cannot be constructed directly by PCR, please see 6.7 and 6.8 for details. 6. Assembly and Fusion To distinguish BBF RFC 53 specific assembly methods based on and from BBF RFC 10 assembly methods, BBF RFC 10 assembly methods SHOULD be called as assembly, use + to separate parts to be assembled, and use - to represent the scar S8/6 after assembly; BBF RFC 53 specific assembly methods SHOULD be called as fusion, use * to separate parts to be fused, and use / to represent the scar S3 after assembly. There are 5 fusion methods based on the fusion product s format: MetaFusion, HeadFusion, TailFusion, HXTFusion, and FullFusion. MetaFusion is RECOMMENDED, because the product will be in the formats with MetaPart, but is used in MetaFusion method, which is more expensive than other enzymes. By using HeadFusion, TailFusion, and FullFusion, the use of is avoid, but the product are not in MetaPart, and parallel assembly [9] is not possible BBF RFC 10 Assembly Not all assembly between any two parts are allowed in BBF RFC 53, since the product MUST meet one of the 10 formats in 5.2. If the two parts both contains MPF or both contains MSF, the assembly is not allowed. Assembly to produce basic formats: Tail + Head = Meta (e.g. BBa_K BBa_K = BBa_K371061) Full + Head = Head (e.g. BBa_B BBa_K = BBa_K371058) Tail + Full = Tail Full + Full = Full Assembly to produce composite formats are listed in MetaFusion M1 * M2 * * MN = M(1/2/ /N) 12

13 Requirements: There are N parts, N>1. Formats of Part1 ~ PartN: Meta or XM or MX or XMX. Part1.=Part2., Part2.=Part3.,, Part(N-1).=PartN.. Any 2 of Part1.~Part(N-1). MUST NOT be the same. If all parts are D-L standard parts, N<4. General method: Part1: Digest with SacI. Add and continue to digest. Gel purify insert. Each of Part2~Part(N-1): Digest with. Gel purify insert. PartN: Digest with. Add SacI and continue to digest. Gel purify vector. Mix purification products in proper ratio and ligate. Examples: BBa_K * BBa_K * BBa_K = BBa_K BBa_K * BBa_K * BBa_K = BBa_K BBa_K * BBa_K * BBa_K = BBa_K BBa_K * BBa_K * BBa_K = BBa_K BBa_K * BBa_K * BBa_K = BBa_K BBa_K * BBa_K * BBa_K = BBa_K

14 Part1 MetaPart1 5'...AGAGGAGCTCTTCA aTGAAGAGATCTACT...3' 3'...TCTCCTCGAGAAGT bACTTCTCTAGATGA...5' SacI PartN MetaPartN 5'...AGAGGAGCTCTTCAoqs...uwyTGAAGAGATCTACT...3' 3'...TCTCCTCGAGAAGTprt...vxzACTTCTCTAGATGA...5' 5'...AGAGGAGCT 3'...TCTCC MetaPart1 MetaSuffix CTTCA aTGAAGAGATCTACT...3' TCGAGAAGT bACTTCTCTAGATGA...5' MetaPartN 5'...AGAGGAGCTCTTCA oqs...uwytgaagagatctact...3' 3'...TCTCCTCGAGAAGTprt...vxzACTTCTCTAGATGA...5' SacI MetaPart1 MetaSuffix 5'...AGAGGAGCT CTTCA aTGAAGAGATCTACT...3' 3'...TCTCC TCGAGAAGT b ACTTCTCTAGATGA...5' MetaPartN MetaSuffix 5'...AGAGGAGCT CTTCA oqs...uwytgaagagatctact...3' 3'...TCTCC TCGAGAAGTprt...vxzACTTCTCTAGATGA...5' Insert Part2~Part(N-1) 5'...AGAGGAGCTCTTCA68a...cegTGAAGAGATCTACT...3'... 3'...TCTCCTCGAGAAGT79b...dfhACTTCTCTAGATGA...5'... MetaPart(N-1) Vector 5'...AGAGGAGCTCTTCA 68a... cegtgaagagatctact...3'... 3'...TCTCCTCGAGAAGT79b...dfh ACTTCTCTAGATGA...5'... MetaPart(N-1) Insert MetaPart1 MetaPart3 ~MetaPart (N-1) MetaPartN MetaSuffix 5'...AGAGGAGCT CTTCA a oqs...uwytgaagagatctact...3' 3'...TCTCC TCGAGAAGT b...dfh......vxzACTTCTCTAGATGA...5' MetaPrefix Ligation MetaPart1/2/.../N MetaPart1 MetaPart(N-1) MetaPartN S3 S3 S3 MetaSuffix 5'...AGAGGAGCTCTTCA a...ceg...oqs...uwyTGAAGAGATCTACT...3' 3'...TCTCCTCGAGAAGT b...dfh...prt...vxzACTTCTCTAGATGA...5' 14

15 6.3. HeadFusion H1 * M2 * * MN = H(1/2/ /N) Requirements: There are N parts, N>1. Formats of Part1: Head or HX. Formats of Part2 ~ PartN: Meta or XM or MX or XMX. Part1.=Part2., Part2.=Part3.,, Part(N-1).=PartN.. Any 2 of Part1.~Part(N-1). MUST NOT be the same. If all parts are D-L standard parts, N<4. General method: Part1: Digest with. Add BglII and continue to digest. Gel purify vector. Each of Part2~Part(N-1): Digest with. Gel purify insert. PartN: (1) or (2). (1) Digest with BglII. Add and continue to digest. Gel purify insert. (2) Double digest with BglII and. Gel purify insert. Mix purification products in proper ratio and ligate. 15

16 Part1 HeadPart1 MetaSuffix 5'...AGAK...024TGAAGAGATCTACT...3' 3'...TCTM...135ACTTCTCTAGATGA...5' PartN MetaPartN 5'...AGAGGAGCTCTTCAoqs...uwyTGAAGAGATCTACT...3' 3'...TCTCCTCGAGAAGTprt...vxzACTTCTCTAGATGA...5' BglII BglII & HeadPart1 MetaSuffix 5'...AGAK TGAAGAGATCTACT...3' 3'...TCTM ACTTCTCTAGATGA...5' BglII MetaPartN MetaPrefix 5'...AGAGGAGCTCTTCAoqs...uwyTGAAGA 3'...TCTCCTCGAGAAGTprt...vxzACTTCTCTAG GATCTACT...3' ATGA...5' HeadPart1 5'...AGAK TGAAGA GATCTACT...3' 3'...TCTM ACTTCTCTAG ATGA...5' MetaPartN MetaPrefix 5'...AGAGGAGCTCTTCA oqs...uwytgaaga GATCTACT...3' 3'...TCTCCTCGAGAAGTprt...vxzACTTCTCTAG ATGA...5' Vector Part2~Part(N-1) MetaPart(N-1) 5'...AGAGGAGCTCTTCA aTGAAGAGATCTACT...3'... 3'...TCTCCTCGAGAAGT bACTTCTCTAGATGA...5'... Insert 5'...AGAGGAGCTCTTCA aTGAAGAGATCTACT...3'... 3'...TCTCCTCGAGAAGT b ACTTCTCTAGATGA...5'... MetaPart(N-1) Insert HeadPart1 MetaPart3 ~MetaPart (N-1) MetaPartN 5'...AGAK oqs...uwytgaaga GATCTACT...3' 3'...TCTM b......vxzACTTCTCTAG ATGA...5' HeadPart1/2/.../N Ligation HeadPart1 MetaPart(N-1) MetaPartN S3 S3 S3 MetaSuffix 5'...AGAK a...oqs...uwyTGAAGAGATCTACT...3' 3'...TCTM b...prt...vxzACTTCTCTAGATGA...5' 16

17 6.4. TailFusion M1 * M2 * * TN = T(1/2/ /N) Requirements: There are N parts, N>1. Formats of Part1 ~ Part(N-1): Meta or XM or MX or XMX. Format of PartN: Tail or XT. Part1.=Part2., Part2.=Part3.,, Part(N-1).=PartN.. Any 2 of Part1.~Part(N-1). MUST NOT be the same. If all parts are D-L standard parts, N<4. General method: Part1: Digest with SacI. Add and continue to digest. Gel purify insert. Each of Part2~Part(N-1): Digest with. Gel purify insert. PartN: Digest with. Add SacI and continue to digest. Gel purify vector. Mix purification products in proper ratio and ligate. 17

18 Part1 MetaPart1 5'...AGAGGAGCTCTTCA aTGAAGAGATCTACT...3' 3'...TCTCCTCGAGAAGT bACTTCTCTAGATGA...5' SacI PartN TailPartN MetaPrefix 5'...AGAGGAGCTCTTCAuwy...TACT...3' 3'...TCTCCTCGAGAAGTvxz...ATGA...5' 5'...AGAGGAGCT 3'...TCTCC MetaPart1 MetaSuffix CTTCA aTGAAGAGATCTACT...3' TCGAGAAGT bACTTCTCTAGATGA...5' MetaPrefix 5'...AGAGGAGCTCTTCA 3'...TCTCCTCGAGAAGTvxz TailPartN uwy...tact...3'...atga...5' SacI MetaPart1 MetaSuffix 5'...AGAGGAGCT CTTCA aTGAAGAGATCTACT...3' 3'...TCTCC TCGAGAAGT b ACTTCTCTAGATGA...5' TailPartN 5'...AGAGGAGCT CTTCA uwy...tact...3' 3'...TCTCC TCGAGAAGTvxz...ATGA...5' Insert Part2~Part(N-1) 5'...AGAGGAGCTCTTCA68a...cegTGAAGAGATCTACT...3'... 3'...TCTCCTCGAGAAGT79b...dfhACTTCTCTAGATGA...5'... MetaPart(N-1) Vector 5'...AGAGGAGCTCTTCA 68a... cegtgaagagatctact...3'... 3'...TCTCCTCGAGAAGT79b...dfh ACTTCTCTAGATGA...5'... MetaPart(N-1) Insert MetaPart1 MetaPart3 ~MetaPart (N-1) TailPartN 5'...AGAGGAGCT CTTCA a uwy...tact...3' 3'...TCTCC TCGAGAAGT b...dfh......ATGA...5' MetaPrefix Ligation TailPart1/2/.../N MetaPart1 MetaPart(N-1) TailPartN S3 S3 S3 5'...AGAGGAGCTCTTCA a...ceg...uwy...TACT...3' 3'...TCTCCTCGAGAAGT b...dfh...vxz...ATGA...5' 18

19 6.5. HXTFusion M1 * M2 * * HXT(N-N) = HXT(N-1/2/ /N) Requirements: There are N parts, N>1. Formats of Part1 ~ Part(N-1): Meta or XM or MX or XMX. Format of PartN: HXT. Part1.=Part2., Part2.=Part3.,, Part(N-1).=PartN.. Any 2 of Part1.~Part(N-1). MUST NOT be the same. If all parts are D-L standard parts, N<4. General method: Part1: Digest with SacI. Add and continue to digest. Gel purify insert. Each of Part2~Part(N-1): Digest with. Gel purify insert. PartN: Digest with. Add SacI and continue to digest. Gel purify vector. Mix purification products in proper ratio and ligate. 19

20 Part1 MetaPart1 5'...AGGAGCTCTTCA aTGAAGAGATCTA...3' 3'...TCCTCGAGAAGT bACTTCTCTAGAT...5' SacI PartN HeadPartN ExtraInfix TailPartN MetaSuffix MetaPrefix 5'...AK...uwyTGAAGAGATCTA...AGGAGCTCTTCAoqs...TA...3' 3'...TM...vxzACTTCTCTAGAT...TCCTCGAGAAGTprt...AT...5' 5'...AGGAGCT 3'...TCC MetaPart1 MetaSuffix CTTCA aTGAAGAGATCTA...3' TCGAGAAGT bACTTCTCTAGAT...5' HeadPartN ExtraInfix MetaSuffix MetaPrefix 5'...AK...uwyTGAAGAGATCTA...AGGAGCTCTTCA 3'...TM...vxzACTTCTCTAGAT...TCCTCGAGAAGTprt TailPartN oqs...ta...3'...at...5' SacI MetaPart1 MetaSuffix 5'...AGGAGCT CTTCA aTGAAGAGATCTA...3' 3'...TCC TCGAGAAGT b ACTTCTCTAGAT...5' HeadPartN MetaSuffix TailPartN 5'...AK...uwyTGAAGAGATCTA...AGGAGCT CTTCA oqs...ta...3' 3'...TM...vxzACTTCTCTAGAT...TCC TCGAGAAGTprt...AT...5' Insert Part2~Part(N-1) MetaPart(N-1) 5'...AGGAGCTCTTCA68a...cegTGAAGAGATCTA...3'... 3'...TCCTCGAGAAGT79b...dfhACTTCTCTAGAT...5'... Vector 5'...AGGAGCTCTTCA 68a... cegtgaagagatcta...3'... 3'...TCCTCGAGAAGT79b...dfh ACTTCTCTAGAT...5'... MetaPart(N-1) Insert HeadPartN MetaSuffix MetaPart1 MetaPart3 ~MetaPart (N-1) TailPartN 5'...AK...uwyTGAAGAGATCTA...AGGAGCT CTTCA a oqs...ta...3' 3'...TM...vxzACTTCTCTAGAT...TCC TCGAGAAGT b...dfh......AT...5' HeadPartN ExtraInfix TailPartN MetaSuffix MetaPrefix S3 S3 S3 5'...AK...uwyTGAAGAGATCTA...AGGAGCTCTTCA a...ceg...oqs...TA...3' 3'...TM...vxzACTTCTCTAGAT...TCCTCGAGAAGT b...dfh...prt...AT...5' Ligation TailPart1/2/.../N MetaPart1 MetaPart(N-1) 20

21 6.6. FullFusion Head-FullFusion H1 * M2 * * TN = F(1/2/ /N) Requirements: There are N parts, N>1. Format of Part1: Head or HX or HXT. Formats of Part2 ~ Part(N-1): Meta or XM or MX or XMX. Format of PartN: Tail or XT or HXT. Part1.=Part2., Part2.=Part3.,, Part(N-1).=PartN.. Any 2 of Part1.~Part(N-1). MUST NOT be the same. If all parts are D-L standard parts, N<4. General method: Part1: Double digest with and PstI. Gel purify vector. Each of Part2~Part(N-1): Digest with. Gel purify insert. PartN: Double digest with and PstI. Gel purify insert. Mix purification products in proper ratio and ligate Tail-FullFusion H1 * M2 * * TN = F(1/2/ /N) Requirements: There are N parts, N>1. Format of Part1: Head or HX or HXT. Formats of Part2 ~ Part(N-1): Meta or XM or MX or XMX. Format of PartN: Tail or XT or HXT. Part1.=Part2., Part2.=Part3.,, Part(N-1).=PartN.. Any 2 of Part1.~Part(N-1). MUST NOT be the same. If all parts are D-L standard parts, N<4. General method: Part1: Double digest with and EcoRI. Gel purify insert. Each of Part2~Part(N-1): Digest with. Gel purify insert. PartN: Double digest with and EcoRI. Gel purify vector. Mix purification products in proper ratio and ligate. 21

22 Part1 HeadPart1 MetaSuffix PstI 5'...AK...024TGAAGAGATCTA...CTGCAG...3' 3'...TM...135ACTTCTCTAGAT...GACGTC...5' PartN TailPartN MetaPrefix Suffix SpeI NotI PstI 5'...AGGAGCTCTTCAuwy...TACTAGTAGCGGCCGCTGCAG...3' 3'...TCCTCGAGAAGTvxz...ATGATCATCGCCGGCGACGTC...5' & PstI & PstI HeadPart1 MetaSuffix 5'...AK TGAAGAGATCTA...CTGCA G...3' 3'...TM ACTTCTCTAGAT...G ACGTC...5' MetaPrefix SpeI NotI 5'...AGGAGCTCTTCA uwy...tactagtagcggccgctgca G...3' 3'...TCCTCGAGAAGTvxz...ATGATCATCGCCGGCG ACGTC...5' TailPartN Vector Part2~Part(N-1) MetaPart(N-1) 5'...AGGAGCTCTTCA aTGAAGAGATCTA...3'... 3'...TCCTCGAGAAGT bACTTCTCTAGAT...5'... Insert 5'...AGGAGCTCTTCA aTGAAGAGATCTA...3'... 3'...TCCTCGAGAAGT b ACTTCTCTAGAT...5'... MetaPart(N-1) Insert HeadPart1 MetaPart3 ~MetaPart (N-1) TailPartN 5'...AK uwy...tactagtagcggccgctgca G...3' 3'...TM b......ATGATCATCGCCGGCG ACGTC...5' SpeI NotI FullPart1/2/.../N Ligation HeadPart1 MetaPart(N-1) TailPartN S3 S3 S3 Suffix SpeI NotI PstI 5'...AK a...uwy...TACTAGTAGCGGCCGCTGCAG...3' 3'...TM b...vxz...ATGATCATCGCCGGCGACGTC...5' 22

23 Part1 Prefix22/20 EcoRI NotI XbaI HeadPart1 & EcoRI MetaSuffix 5'...GAATTCGCGGCCGCTTCTAGAK...024TGAAGAGATCTA...3' 3'...CTTAAGCGCCGGCGAAGATCTM...135ACTTCTCTAGAT...5' PartN TailPartN MetaPrefix EcoRI 5'...GAATTC...AGGAGCTCTTCAuwy...TA...3' 3'...CTTAAG...TCCTCGAGAAGTvxz...AT...5' & EcoRI HeadPart1 MetaSuffix NotI XbaI 5'...G AATTCGCGGCCGCTTCTAGAK TGAAGAGATCTA...3' 3'...CTTAA GCGCCGGCGAAGATCTM ACTTCTCTAGAT...5' MetaPrefix 5'...G AATTC...AGGAGCTCTTCA uwy...ta...3' 3'...CTTAA G...TCCTCGAGAAGTvxz...AT...5' TailPartN Insert Part2~Part(N-1) 5'...AGGAGCTCTTCA aTGAAGAGATCTA...3'... 3'...TCCTCGAGAAGT bACTTCTCTAGAT...5'... MetaPart(N-1) Vector 5'...AGGAGCTCTTCA aTGAAGAGATCTA...3'... 3'...TCCTCGAGAAGT b ACTTCTCTAGAT...5'... MetaPart(N-1) Insert HeadPart1 MetaPart3 ~MetaPart (N-1) TailPartN NotI XbaI 5'...G AATTCGCGGCCGCTTCTAGAK uwy...ta...3' 3'...CTTAA GCGCCGGCGAAGATCTM b......AT...5' Ligation FullPart1/2/.../N HeadPart1 MetaPart(N-1) TailPartN Prefix22/20 S3 S3 S3 EcoRI NotI XbaI 5'...GAATTCGCGGCCGCTTCTAGAK a...uwy...TA...3' 3'...CTTAAGCGCCGGCGAAGATCTM b...vxz...AT...5' 23

24 HXT-FullFusion HXT(1-1) * M2 * * MN = F(1/2/ /N/1) Requirements: There are N parts, N>0. Format of Part1: HXT. Formats of Part2 ~ PartN: Meta or XM or MX or XMX. Part1.=Part2., Part2.=Part3.,, Part(N-1).=PartN., PartN.=Part1.. Any 2 of Part1.~PartN. MUST NOT be the same. If all parts are D-L standard parts, N<3. General method: Part1: Digest with. Gel purify vector. Each of Part2~PartN: Digest with. Gel purify insert. Mix purification products in proper ratio and ligate. Examples: BBa_K (similar to HXT) * BBa_K BBa_K (similar to HXT) * BBa_K BBa_K (similar to HXT) * BBa_K BBa_K (similar to HXT) * BBa_K BBa_K (similar to HXT) * BBa_K BBa_K (similar to HXT) * BBa_K

25 Part1 MetaSuffix MetaPrefix 5'...AK...024TGAAGAGATCTA...AGGAGCTCTTCAuwy...TA...3' 3'...TM...135ACTTCTCTAGAT...TCCTCGAGAAGTvxz...AT...5' HeadPart1 HeadPart1 ExtraInfix ExtraInfix MetaSuffix MetaPrefix TailPart1 TailPart1 5'...AK TGAAGAGATCTA...AGGAGCTCTTCA uwy...ta...3' 3'...TM ACTTCTCTAGAT...TCCTCGAGAAGTvxz...AT...5' Vector Part2~PartN MetaPartN 5'...AGGAGCTCTTCA aTGAAGAGATCTA...3'... 3'...TCCTCGAGAAGT bACTTCTCTAGAT...5'... 5'...AGGAGCTCTTCA aTGAAGAGATCTA...3'... 3'...TCCTCGAGAAGT b ACTTCTCTAGAT...5'... MetaPartN Insert HeadPart1 MetaPart3 ~MetaPart N TailPart1 5'...AK uwy...ta...3' 3'...TM b......AT...5' Ligation FullPart1/2/.../N/1 HeadPart1 MetaPartN TailPart1 S3 S3 S3 5'...AK a...uwy...TA...3' 3'...TM b...vxz...AT...5' 25

26 6.7. Short Parts and de novo DNA synthesis Some parts are very short, such as stop codon - RBS, tag, linker, cleavage site. Construction of plasmids for these parts is usually not necessary.[10] They can be constructed from chemically synthesized oligos and used for assembly and fusion directly. Different methods can be used according to the length of the part. If the fragment needed is shorter than 60bp after digest (from cohesive end to cohesive end), the 2 oligos can be design to be the same as the 2 strands with cohesive ends. The oligos can be annealed and directly used for assembly or fusion. Annealing of oligos are very easy, just heat to 94 C, cool to room temperature and used for ligation. The annealed oligos should be stored at -20 C as aliquots. If the fragment including the cutting sites are shorter than 100bp, primer annealing and extension can be used. Synthesize 2 oligos with ~20bp overlap, the 5 end should include the cutting site and extra bases according to the instruction of the enzyme. Do PCR without template for 3 cycles, digest, purify the product, and used for ligation. If the fragment from cohesive end to cohesive end is shorter than 200bp, each of the two strands can be divided into oligos shorter than 60nt, and keep each annealing fragments to have similar annealing temperatures. These oligos can be mixed, phosphorylated by T4PNK, annealed and used for ligation. The less the number of oligos, the larger the possibility of success. For longer parts, de novo DNA synthesis are also useful, when there is no template for PCR or the sequence need to be optimized. Assembly Standard 53 is a scarless assembly method, so the problem of long part synthesis can be easily converted to the synthesis of short parts using methods above, and the fusion of the parts. 1. Design the sequence of the final part needed. 2. Choose each S3 site every 150~200bp. Try to choose all the S3 s with different sequences, so that they can be fused in a single ligation reaction. If this is not possible, divide the whole part into smaller parts first, until it is possible in each smaller parts. 3. According to the format of the needed part and S3 s, choose a fusion strategy that can get each of the smaller parts first, and the whole part at last. 4. Synthesis each part separated by S3 s using the method above. 5. Construct the final part according to the fusion strategy Site-Directed Mutagenesis 1. Design the sequence of the final part needed. Mark the mutation sites. 2. For each mutation site, an S3 is need, the site SHOULD be in the S3 or near the S3. However, if 2 or more sites are closer than 50bp, these sites MAY share a single S3 in the center of them. 26

27 3. Try to choose all the S3 s with different sequences, so that they can be fused in a single ligation reaction. If this is not possible, divide the whole part into smaller parts first, until it is possible in each smaller parts. 4. According to the format of the needed part and S3 s, choose a fusion strategy that can get each of the smaller parts first, and the whole part at last. 5. Use a primer design software to design primers for each part separated by S3 s. The S3 SHOULD be fixed as the 5 end of the primer. Add cutting sites to the 5 end of each primer. Add extra bases according to the instruction of the enzymes. 6. Use the primers to amplify each parts, and construct the final part according to the fusion strategy. 7. Author s Contact Information Hao Jiang: haojiang@mail.ustc.edu.cn Yang Zhang: zocean@mail.ustc.edu.cn Ruijun Zhu: rjchu@mail.ustc.edu.cn Chang Liu: lctj@mail.ustc.edu.cn Duo An: anduo@mail.ustc.edu.cn Ying Zhang: praying@mail.ustc.edu.cn Ge Gao: singhigh@mail.ustc.edu.cn Jing Yang: yangjing@mail.ustc.edu.cn References [1] s. [2] Che, A. (2004). "BioBricks++: Simplifying Assembly of Standard DNA Components." [3] Won, J. I. and A. E. Barron (2002). "A new cloning method for the preparation of long repetitive polypeptides without a sequence requirement." Macromolecules 35(22): [4] Chu, H. S., C. Y. Choi, et al. (2008). "Analysis and comparison of cloning methods for the preparation of repetitive polypeptides." Korean Journal of Chemical Engineering 25(5): [5] Meyer, M. M., K. Hiraga, et al. (2006). "Combinatorial recombination of gene fragments to construct a library of chimeras." Current Protocols in Protein Science Chapter 26: Unit [6] Engler, C., R. Kandzia, et al. (2008). "A One Pot, One Step, Precision Cloning 27

28 Method with High Throughput Capability." Plos One 3(11). [7] Engler, C., R. Gruetzner, et al. (2009). "Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes." Plos One 4(5). [8] [9] [10] 28