Golden Gate Cloning

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Golden Gate assembly involves digesting DNA sequences containing a type IIS restriction enzyme cut site and ligating them together. Golden Gate assembly.svg
Golden Gate assembly involves digesting DNA sequences containing a type IIS restriction enzyme cut site and ligating them together.

Golden Gate Cloning or Golden Gate assembly [1] is a molecular cloning method that allows a researcher to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIS restriction enzymes and T4 DNA ligase. [2] This assembly is performed in vitro . Most commonly used Type IIS enzymes include BsaI, BsmBI, and BbsI.

Contents

Unlike standard Type II restriction enzymes like EcoRI and BamHI, these enzymes cut DNA outside of their recognition sites and, therefore, can create non-palindromic overhangs. [3] Since 256 potential overhang sequences are possible, multiple fragments of DNA can be assembled by using combinations of overhang sequences. [3] In practice, this means that Golden Gate Cloning is typically scarless. Additionally, because the final product does not have a Type IIS restriction enzyme recognition site, the correctly-ligated product cannot be cut again by the restriction enzyme, meaning the reaction is essentially irreversible. [3] This has multiple benefits, the first is that it is possible to do digestion and ligation of the DNA fragments in a single reaction, in contrast to conventional cloning methods where these reactions are separate. The second is higher efficiency [1] because the end product cannot be cut again by the restriction enzyme.

A typical thermal cycler protocol oscillates between 37 °C (optimal for restriction enzymes) and 16 °C (optimal for ligases) many times. [4] While this technique can be used for a single insert, researchers have used Golden Gate Cloning to assemble many pieces of DNA simultaneously. [5]

Seamless cloning

Scar sequences are common in multiple segment DNA assembly. In the multisegment assembly method Gateway, segments are added into the donor with additional ATT sequences, which overlap in those added segments, and this results in the segments separated by the ATT sequences. [6] In BioBrick assembly, an eight-nucleotide scar sequence, which codes for a tyrosine and a stop codon, is left between every segment added into the plasmid. [6]

Golden Gate assembly uses Type IIS restriction enzymes cutting outside their recognition sequences. [6] Also, the same Type IIS restriction enzyme can generate copious different overhangs on the inserts and the vector; for instance, BsaI creates 256 four-basepair overhangs. [6] If the overhangs are carefully designed, the segments are ligated without scar sequences between them, and the final construct can be quasi-scarless, where the restriction enzyme sites remain on both sides of the insert. [6] As additional segments can be inserted into the vectors without scars within an open reading frame, Golden Gate is widely used in protein engineering. [6]

Plasmid design

Although Golden Gate Cloning speeds up multisegment cloning, careful design of donor and recipient plasmids is required. [5] Scientists at New England Biolabs have successfully demonstrated the assembly of 35 fragments via a single-tube Golden Gate Assembly reaction. [7] Critical to this method of assembly, the vector backbone of the destination plasmid and all the assembly fragments are flanked by Type IIS restriction enzyme recognition sites, as this subtype of restriction enzymes cut downstream from their recognition sites. After cutting, each assembly active piece of DNA has unique overhangs that anneal to the next fragment of DNA in the planned assembly and become ligated, building the assembly. While it is also possible for an overhang to anneal back to its original complementary overhang associated with the upstream recognition site and become ligated, re-forming the original sequence, this will be susceptible to further cutting throughout the assembly reaction.

Cloning standards

Restriction enzyme DNA assembly has cloning standards to minimize the change in cloning efficiency and the function of the plasmid, which can be caused by compatibility of the restriction sites on the insert and those on the vector. [8]

Golden Gate assembly's cloning standards have two tiers. [8] First-tier Golden Gate assembly constructs the single-gene construct by adding in genetic elements such as promoter, open reading frames, and terminators. [8] Then, second-tier Golden Gate assembly combine several constructs made in first-tier assembly to make a multigene construct. [8] To achieve second-tier assembly, modular cloning (MoClo) system and GoldenBraid2.0 standard are used. [8]

MoClo system

Schematic workflow for generating complex combinatorial DNA libraries Schematic workflow for generating complex combinatorial DNA libraries.svg
Schematic workflow for generating complex combinatorial DNA libraries

Modular Cloning, or MoClo, is an assembly method introduced in 2011 by Ernst Weber et al., whereby using Type IIS restriction sites, the user can ligate at least six DNA parts together into a backbone in a one-pot reaction. It is a method based on Golden Gate Assembly, where Type IIS restriction enzymes cleave outside of their recognition site to one side, allowing for removal of those restriction sites from the design. This helps eliminate excess base pairs, or scars, from forming between DNA Parts. However, in order to ligate together properly, MoClo utilizes a set of 4-base pair fusion sites, which remain between parts after ligation, forming 4-base pair scars between DNA parts in the final DNA sequence following ligation of two or more parts. [9]

MoClo utilizes a parallel approach, where all constructs from tier-one(level 0 modules) have restriction sites for BpiI on both sides of the inserts. The vector(also known as "destination vector"), where genes will be added, has an outward-facing BsaI restriction site with a drop-out screening cassette. [8] LacZ is a common screening cassette, where it is replaced by the multigene construct on the destination vector. [8] Each tier-one construct and the vector have different overhangs on them yet complementary to the overhang of the next segment, and this determines the layout of the final multigene construct. [8]  Golden Gate Cloning usually starts with level 0 modules. [5] However, if the level 0 module is too large, cloning will start from level -1 fragments, which have to be sequenced, to help cloning the large construct. [5] If starting from level -1 fragments, the level 0 modules do not need to be sequenced again, whereas if starting from level 0 modules, the modules must be sequenced. [5]

Level 0 modules

Level 0 modules are the base for MoClo system, where they contain genetic elements like a promoter, a 5' untranslated region (UTR), a coding sequence, and a terminator. [5] For the purpose of Golden Gate Cloning, the internal sequences of level 0 modules should not contain type IIS restriction enzymes sites for BsaI, BpiI, and Esp3I while surrounded by two BsaI restriction sites in inverted orientation. [5] Level 0 modules without type IIS restriction sites flanking can add the BsaI sites during the process of Golden Gate Cloning. [5]

If the level 0 modules contains any unwanted restriction site, they can be mutated in silico by removing one nucleotide from the Type IIS restriction site. [5] In this process, one needs to make sure that the introduced mutation will not affect the genetic function encoded by the sequence of interest. [5] A silent mutation in the coding sequence is preferred, for it neither changes the protein sequence nor the function of the gene of interest. [5]

Level -1 fragments

Level -1 fragments are used to help cloning large level 0 modules. [5] To clone level -1 fragments, blunt-end cloning with restriction ligation can be used. [5] The vector used in cloning level -1 fragments cannot contain Type IIS restriction site BpiI that is used for the following assembly step. [5] Moreover, the vector should also have a different selection marker from the destination vector in next assembly step, for example, if spectinomycin resistance is used in level 0 modules, level -1 fragments should have another antibiotic resistance like ampicillin. [5]  

Level 1 constructs

The level 1 destination vector determines the position and orientation of each gene in the final construct. [10] There are fourteen available level 1 vectors, which differ only by the sequence of the flanking fusion sites while being identical in the internal fusion sites. [10] Hence, all vectors can assemble the same level 0 parts. [10]

As all level 1 vectors are binary plasmids, they are used for Agrobacterium mediated temporary expression in plants. [10]

Level 2 constructs

Level 2 vectors have two inverted BpiI sites from the insertion of level 1 modules. [10] The upstream fusion site is compatible to a gene cloned in level 1 vector while the downstream fusion site has a universal sequence. [10] Each cloning allows 2-6 genes to be inserted in the same vector. [10]

Adding more genes in one cloning step is not recommended, for this would result in incorrect constructs. [10] On one hand, this can induce more restriction sites in the construct, where this open construct allows additional genes be added. [10] On the other hand, this can also eliminate restriction sites, where this close construct stop the further addition of genes. [10]

Therefore, constructs of more than six genes need successive cloning steps, which requires end-linkers containing BsaI or BsmBI internal restriction sites and blue or purple markers. [10] Each cloning step needs to alternate the restriction site and the marker. [10] Furthermore, two restriction enzymes are needed, where BpiI is used for releasing level 1 modules from level 1 constructs and BsaI/BsmBI is for digesting and opening the recipient level 2-n plasmid. [10] When screening, the correct colonies should alternate from blue to purple every cloning step, but if a "closed" end-linker is used, the colonies will be white. [10]  

Level M constructs

Level M vectors are similar to level 2 vectors, but have a BsaI site located upstream of the two inverted BpiI sites. [11] When one or several genes are cloned in a level M vector, a second BsaI is added at the end of the construct via a Level M end-linker (ref). This allows a fragment containing all assembled genes to be excised from the vector and subcloned in a next level of cloning (Level P).

Level P constructs

Level P vectors are similar to level M constructs except that the BpiI sites are replaced by BsaI sites and the BsaI sites are replaced by BpiI sites. Several level M constructs with compatible fusion sites can be subcloned into a level P vector in one step. Theoretically, as many as 36 genes can be assembled in one construct using 6 parallel level M reactions (each required for assembly of 6 genes per level M construct) followed by one final level P reaction. In practice, fewer genes are usually assembled as most cloning projects do not require so many genes. The structure of level M and P vectors is designed in a such as way that genes cloned in level P constructs can be further assembled in level M vectors. Repeated cloning in level M and P vectors forms a loop that can be repeated indefinitely to assemble progressively large constructs.

GoldenBraid

In standard Golden Gate Cloning, the restriction sites from the previous tier construct cannot be reused. [12] To add more genes to the construct, restriction sites of a different Type IIS restriction enzyme need to be added to the destination vector. [12] This can be done using either level 2, or M and P. A variant version of level M and P is also provided by GoldenBraid.

GoldenBraid overcomes the problem of designing numerous destination vectors by having a double loop, which is the "braid," to allow binary assembly of multiple constructs. [12] There are two levels of destination plasmids, level α and level Ω. [12] Each level of plasmids can be used as entry plasmids for the other level of plasmids for multiple times because both levels of plasmids have different Type IIS restriction sites that are in inverted orientation. [12] For counterselection, the two levels of plasmids differ in their antibiotic resistance markers. [12]

Golden mutagenesis

The Golden Gate Cloning principle can also be applied to perform mutagenesis termed Golden Mutagenesis. The technology is easy to implement as a web tool is available for primer design (https://msbi.ipb-halle.de/GoldenMutagenesisWeb/) and the vectors are deposited at addgene (http://www.addgene.org/browse/article/28196591/). [13]

Name

The name Golden Gate Assembly comes from a proposal of Yuri Gleba. [1] It shall refer on the one hand to the Gateway Technology, on the other hand picture the higher precision with a bridge connecting the streets of two shores seamlessly. One of the most well known bridges is the Golden Gate Bridge in San Francisco.

Related Research Articles

Protein engineering is the process of developing useful or valuable proteins through the design and production of unnatural polypeptides, often by altering amino acid sequences found in nature. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles. It has been used to improve the function of many enzymes for industrial catalysis. It is also a product and services market, with an estimated value of $168 billion by 2017.

<span class="mw-page-title-main">Cloning vector</span> Small piece of maintainable DNA

A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium. The vector contains features that allow for the convenient insertion of a DNA fragment into the vector or its removal from the vector, for example through the presence of restriction sites. The vector and the foreign DNA may be treated with a restriction enzyme that cuts the DNA, and DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and vector DNA and foreign DNA with compatible ends can then be joined by molecular ligation. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.

<span class="mw-page-title-main">Library (biology)</span>

In molecular biology, a library is a collection of DNA fragments that is stored and propagated in a population of micro-organisms through the process of molecular cloning. There are different types of DNA libraries, including cDNA libraries, genomic libraries and randomized mutant libraries. DNA library technology is a mainstay of current molecular biology, genetic engineering, and protein engineering, and the applications of these libraries depend on the source of the original DNA fragments. There are differences in the cloning vectors and techniques used in library preparation, but in general each DNA fragment is uniquely inserted into a cloning vector and the pool of recombinant DNA molecules is then transferred into a population of bacteria or yeast such that each organism contains on average one construct. As the population of organisms is grown in culture, the DNA molecules contained within them are copied and propagated.

A cosmid is a type of hybrid plasmid that contains a Lambda phage cos sequence. They are often used as a cloning vector in genetic engineering. Cosmids can be used to build genomic libraries. They were first described by Collins and Hohn in 1978. Cosmids can contain 37 to 52 kb of DNA, limits based on the normal bacteriophage packaging size. They can replicate as plasmids if they have a suitable origin of replication (ori): for example SV40 ori in mammalian cells, ColE1 ori for double-stranded DNA replication, or f1 ori for single-stranded DNA replication in prokaryotes. They frequently also contain a gene for selection such as antibiotic resistance, so that the transformed cells can be identified by plating on a medium containing the antibiotic. Those cells which did not take up the cosmid would be unable to grow.

A DNA construct is an artificially-designed segment of DNA borne on a vector that can be used to incorporate genetic material into a target tissue or cell. A DNA construct contains a DNA insert, called a transgene, delivered via a transformation vector which allows the insert sequence to be replicated and/or expressed in the target cell. This gene can be cloned from a naturally occurring gene, or synthetically constructed. The vector can be delivered using physical, chemical or viral methods. Typically, the vectors used in DNA constructs contain an origin of replication, a multiple cloning site, and a selectable marker. Certain vectors can carry additional regulatory elements based on the expression system involved.

A restriction digest is a procedure used in molecular biology to prepare DNA for analysis or other processing. It is sometimes termed DNA fragmentation, though this term is used for other procedures as well. In a restriction digest, DNA molecules are cleaved at specific restriction sites of 4-12 nucleotides in length by use of restriction enzymes which recognize these sequences.

<span class="mw-page-title-main">Multiple cloning site</span>

A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. The purpose of an MCS in a plasmid is to allow a piece of DNA to be inserted into that region.

A genomic library is a collection of overlapping DNA fragments that together make up the total genomic DNA of a single organism. The DNA is stored in a population of identical vectors, each containing a different insert of DNA. In order to construct a genomic library, the organism's DNA is extracted from cells and then digested with a restriction enzyme to cut the DNA into fragments of a specific size. The fragments are then inserted into the vector using DNA ligase. Next, the vector DNA can be taken up by a host organism - commonly a population of Escherichia coli or yeast - with each cell containing only one vector molecule. Using a host cell to carry the vector allows for easy amplification and retrieval of specific clones from the library for analysis.

<span class="mw-page-title-main">Blue–white screen</span> DNA screening technique

The blue–white screen is a screening technique that allows for the rapid and convenient detection of recombinant bacteria in vector-based molecular cloning experiments. This method of screening is usually performed using a suitable bacterial strain, but other organisms such as yeast may also be used. DNA of transformation is ligated into a vector. The vector is then inserted into a competent host cell viable for transformation, which are then grown in the presence of X-gal. Cells transformed with vectors containing recombinant DNA will produce white colonies; cells transformed with non-recombinant plasmids grow into blue colonies.

<i>Hae</i>III Enzyme

HaeIII is one of many restriction enzymes (endonucleases) a type of prokaryotic DNA that protects organisms from unknown, foreign DNA. It is a restriction enzyme used in molecular biology laboratories. It was the third endonuclease to be isolated from the Haemophilus aegyptius bacteria. The enzyme's recognition site—the place where it cuts DNA molecules—is the GGCC nucleotide sequence which means it cleaves DNA at the site 5′-GG/CC-3. The recognition site is usually around 4-8 bps.This enzyme's gene has been sequenced and cloned. This is done to make DNA fragments in blunt ends. HaeIII is not effective for single stranded DNA cleavage.

<span class="mw-page-title-main">BioBrick</span> Standard for components used in DNA synthesis

BioBrick parts are DNA sequences which conform to a restriction-enzyme assembly standard. These building blocks are used to design and assemble larger synthetic biological circuits from individual parts and combinations of parts with defined functions, which would then be incorporated into living cells such as Escherichia coli cells to construct new biological systems. Examples of BioBrick parts include promoters, ribosomal binding sites (RBS), coding sequences and terminators.

Artificial gene synthesis, or simply gene synthesis, refers to a group of methods that are used in synthetic biology to construct and assemble genes from nucleotides de novo. Unlike DNA synthesis in living cells, artificial gene synthesis does not require template DNA, allowing virtually any DNA sequence to be synthesized in the laboratory. It comprises two main steps, the first of which is solid-phase DNA synthesis, sometimes known as DNA printing. This produces oligonucleotide fragments that are generally under 200 base pairs. The second step then involves connecting these oligonucleotide fragments using various DNA assembly methods. Because artificial gene synthesis does not require template DNA, it is theoretically possible to make a completely synthetic DNA molecule with no limits on the nucleotide sequence or size.

NdeI is an endonuclease isolated from Neisseria denitrificans.

Topoisomerase-based cloning is a molecular biology technique in which DNA fragments are cloned into specific vectors without the requirement for DNA ligases. Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3'-end of the PCR products. This characteristic is exploited in "sticky end" TOPO TA cloning. For "blunt end" TOPO cloning, the recipient vector does not have overhangs and blunt-ended DNA fragments can be cloned.

The Gateway cloning method, invented and commercialized by Invitrogen since the late 1990s, is the cloning method of the integration and excision recombination reactions that take place when bacteriophage lambda infects bacteria. This technology provides a fast and highly efficient way to transport DNA sequences into multi-vector systems for functional analysis and protein expression using Gateway att sites, and two proprietary enzyme mixes called BP Clonase and LR Clonase. In vivo, these recombination reactions are facilitated by the recombination of attachment sites from the lambda/phage chromosome (attP) and the bacteria (attB). As a result of recombination between the attP and attB sites, the phage integrates into the bacterial genome flanked by two new recombination sites. The removal of the phage from the bacterial chromosome and the regeneration of attP and attB sites can both result from the attL and attR sites recombining under specific circumstances.

Paired-end tags (PET) are the short sequences at the 5’ and 3' ends of a DNA fragment which are unique enough that they (theoretically) exist together only once in a genome, therefore making the sequence of the DNA in between them available upon search or upon further sequencing. Paired-end tags (PET) exist in PET libraries with the intervening DNA absent, that is, a PET "represents" a larger fragment of genomic or cDNA by consisting of a short 5' linker sequence, a short 5' sequence tag, a short 3' sequence tag, and a short 3' linker sequence. It was shown conceptually that 13 base pairs are sufficient to map tags uniquely. However, longer sequences are more practical for mapping reads uniquely. The endonucleases used to produce PETs give longer tags but sequences of 50–100 base pairs would be optimal for both mapping and cost efficiency. After extracting the PETs from many DNA fragments, they are linked (concatenated) together for efficient sequencing. On average, 20–30 tags could be sequenced with the Sanger method, which has a longer read length. Since the tag sequences are short, individual PETs are well suited for next-generation sequencing that has short read lengths and higher throughput. The main advantages of PET sequencing are its reduced cost by sequencing only short fragments, detection of structural variants in the genome, and increased specificity when aligning back to the genome compared to single tags, which involves only one end of the DNA fragment.

<span class="mw-page-title-main">Molecular cloning</span> Set of methods in molecular biology


Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.

<span class="mw-page-title-main">In vitro recombination</span> Process of isolation and amplification of DNA segments

Recombinant DNA (rDNA), or molecular cloning, is the process by which a single gene, or segment of DNA, is isolated and amplified. Recombinant DNA is also known as in vitro recombination. A cloning vector is a DNA molecule that carries foreign DNA into a host cell, where it replicates, producing many copies of itself along with the foreign DNA. There are many types of cloning vectors such as plasmids and phages. In order to carry out recombination between vector and the foreign DNA, it is necessary the vector and DNA to be cloned by digestion, ligase the foreign DNA into the vector with the enzyme DNA ligase. And DNA is inserted by introducing the DNA into bacteria cells by transformation.

<span class="mw-page-title-main">Jumping library</span>

Jumping libraries or junction-fragment libraries are collections of genomic DNA fragments generated by chromosome jumping. These libraries allow the analysis of large areas of the genome and overcome distance limitations in common cloning techniques. A jumping library clone is composed of two stretches of DNA that are usually located many kilobases away from each other. The stretch of DNA located between these two "ends" is deleted by a series of biochemical manipulations carried out at the start of this cloning technique.

<span class="mw-page-title-main">Ligation (molecular biology)</span>

Ligation is the joining of two nucleic acid fragments through the action of an enzyme. It is an essential laboratory procedure in the molecular cloning of DNA, whereby DNA fragments are joined to create recombinant DNA molecules (such as when a foreign DNA fragment is inserted into a plasmid). The ends of DNA fragments are joined by the formation of phosphodiester bonds between the 3'-hydroxyl of one DNA terminus with the 5'-phosphoryl of another. RNA may also be ligated similarly. A co-factor is generally involved in the reaction, and this is usually ATP or NAD+. Eukaryotic cells ligases belong to ATP type, and NAD+ - dependent are found in bacteria (e.g. E. coli).

References

  1. 1 2 3 Engler, Carola; Kandzia, Romy; Marillonnet, Sylvestre (2008-11-05). "A One Pot, One Step, Precision Cloning Method with High Throughput Capability". PLOS ONE. 3 (11): e3647. Bibcode:2008PLoSO...3.3647E. doi: 10.1371/journal.pone.0003647 . ISSN   1932-6203. PMC   2574415 . PMID   18985154.
  2. ↑ Biolabs, New England. "Golden Gate Assembly | NEB". www.neb.com. Retrieved 2017-04-26.
  3. 1 2 3 Weber, Ernst; Engler, Carola; Gruetzner, Ramona; Werner, Stefan; Marillonnet, Sylvestre (2011-02-18). "A Modular Cloning System for Standardized Assembly of Multigene Constructs". PLOS ONE. 6 (2): e16765. Bibcode:2011PLoSO...616765W. doi: 10.1371/journal.pone.0016765 . ISSN   1932-6203. PMC   3041749 . PMID   21364738.
  4. ↑ Engler, Carola; Gruetzner, Ramona; Kandzia, Romy; Marillonnet, Sylvestre (2009-05-14). "Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes". PLOS ONE. 4 (5): e5553. Bibcode:2009PLoSO...4.5553E. doi: 10.1371/journal.pone.0005553 . ISSN   1932-6203. PMC   2677662 . PMID   19436741.
  5. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Engler, Carola; Marillonnet, Sylvestre (2014-01-01). Golden Gate cloning. Methods in Molecular Biology. Vol. 1116. pp. 119–131. doi:10.1007/978-1-62703-764-8_9. ISBN   978-1-62703-763-1. ISSN   1940-6029. PMID   24395361.
  6. 1 2 3 4 5 6 Sands, Bryan; Brent, Roger (2016-01-01). "Overview of post Cohen-Boyer methods for single segment cloning and for multisegment DNA assembly". Current Protocols in Molecular Biology. 113 (1): 3.26.1–3.26.20. doi:10.1002/0471142727.mb0326s113. ISSN   1934-3647. PMC   4853029 . PMID   27152131.
  7. ↑ Pryor, John M.; Potapov, Vladimir; Kucera, Rebecca B.; Bilotti, Katharina; Cantor, Eric J.; Lohman, Gregory J. S. (2020-09-02). "Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design". PLOS ONE. 15 (9): e0238592. Bibcode:2020PLoSO..1538592P. doi: 10.1371/journal.pone.0238592 . ISSN   1932-6203. PMC   7467295 . PMID   32877448.
  8. 1 2 3 4 5 6 7 8 Casini, Arturo; Storch, Marko; Baldwin, Geoffrey S.; Ellis, Tom (2015). "Bricks and blueprints: methods and standards for DNA assembly" (PDF). Nature Reviews. Molecular Cell Biology. 16 (9): 568–576. doi:10.1038/nrm4014. hdl: 10044/1/31281 . ISSN   1471-0080. PMID   26081612. S2CID   3502437.
  9. ↑ "Team:BostonU/MoClo - 2014.igem.org". 2014.igem.org. Retrieved 2021-09-11.
  10. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Marillonnet, Sylvestre; Werner, Stefan (2015-01-01). Assembly of Multigene Constructs Using Golden Gate Cloning. Methods in Molecular Biology. Vol. 1321. pp. 269–284. doi:10.1007/978-1-4939-2760-9_19. ISBN   978-1-4939-2759-3. ISSN   1940-6029. PMID   26082229.
  11. ↑ Werner S, Engler C, Weber E, Gruetzner R, Marillonnet S. Bioeng Bugs. 2012 Jan 1;3(1):38-43.
  12. 1 2 3 4 5 6 Sarrion-Perdigones, Alejandro; Falconi, Erica Elvira; Zandalinas, Sara I.; Juárez, Paloma; Fernández-del-Carmen, Asun; Granell, Antonio; Orzaez, Diego (2011-07-07). "GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules". PLOS ONE. 6 (7): e21622. Bibcode:2011PLoSO...621622S. doi: 10.1371/journal.pone.0021622 . ISSN   1932-6203. PMC   3131274 . PMID   21750718.
  13. ↑ Pascal Püllmann, Chris Ulpinnis, Sylvestre Marillonnet, Ramona Gruetzner, Steffen Neumann (2019-07-29), "Golden Mutagenesis: An efficient multi-site-saturation mutagenesis approach by Golden Gate cloning with automated primer design", Scientific Reports (in German), vol. 9, no. 1, pp. 1–11, Bibcode:2019NatSR...910932P, doi:10.1038/s41598-019-47376-1, ISSN   2045-2322, PMC   6662682 , PMID   31358887 {{citation}}: CS1 maint: multiple names: authors list (link)
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