Combinatorial Recombination of Gene Fragments to Construct a Library of Chimeras

Farrow, Mary F.; Arnold, Frances H.

doi:10.1002/0471140864.ps2602s44

Cited by 8 publications

(4 citation statements)

References 25 publications

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“…The use of Type IIs enzymes for DNA shuffling has been reported before [17], [18], [19], [20], [28]. However, assembly of the modules was performed from gel purified pre-digested DNA fragments.…”

Section: Discussionmentioning

confidence: 99%

“…Using restriction enzymes would have several advantages such as providing the ability to shuffle genes irrespective of their degree of homology, providing flexibility and control regarding the number of recombination events in each shuffled gene, and the ability to shuffle very large genes or several regions within large genes (independence from PCR amplification). In fact, two DNA shuffling strategies have earlier been developed based on the use of type IIB or type IIs restriction enzymes [17] , [18] , [19] , [20] . However, these protocols are quite complex to perform, require several successive steps, and in many cases still rely on PCR for amplification of the library since only small amount of recombinant templates is obtained.…”

Section: Introductionmentioning

confidence: 99%

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Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes

Engler¹,

Gruetzner²,

Kandzia³

et al. 2009

PLoS ONE

930

872

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We have developed a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones obtained containing the desired construct. This protocol is based on the use of type IIs restriction enzymes and is performed by simply subjecting a mix of 10 undigested input plasmids (nine insert plasmids and the acceptor vector) to a restriction-ligation and transforming the resulting mix in competent cells. The efficiency of this protocol allows generating libraries of recombinant genes by combining in one reaction several fragment sets prepared from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three parental templates (bovine cationic trypsinogen, bovine anionic trypsinogen and human cationic trypsinogen) each divided in 9 separate modules. We show that one round of shuffling using the 27 trypsinogen entry plasmids can easily produce the 19,683 different possible combinations in one single restriction-ligation and that expression screening of a subset of the library allows identification of variants that can lead to higher expression levels of trypsin activity. This protocol, that we call ‘Golden Gate shuffling’, is robust, simple and efficient, can be performed with templates that have no homology, and can be combined with other shuffling protocols in order to introduce any variation in any part of a given gene.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes

Engler¹,

Gruetzner²,

Kandzia³

et al. 2009

PLoS ONE

930

872

View full text Add to dashboard Cite

show abstract

“…47 b-lactamase may also be put to productive use, for example, in anti-cancer ADEPT therapies 13 and in measuring gene expression levels in cells. 48 b-lactamases have been widely used as model systems in the development of combinatorial library construction methods 29,49 due to inexpensive, high-throughput activity screens. blactamase libraries can provide insights on hydrolysis activity of b-lactam derivatives.…”

Section: Focused Designsmentioning

confidence: 99%

Structure‐based design of combinatorial mutagenesis libraries

2015

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The development of protein variants with improved properties (thermostability, binding affinity, catalytic activity, etc.) has greatly benefited from the application of high-throughput screens evaluating large, diverse combinatorial libraries. At the same time, since only a very limited portion of sequence space can be experimentally constructed and tested, an attractive possibility is to use computational protein design to focus libraries on a productive portion of the space. We present a general-purpose method, called "Structure-based Optimization of Combinatorial Mutagenesis" (SOCoM), which can optimize arbitrarily large combinatorial mutagenesis libraries directly based on structural energies of their constituents. SOCoM chooses both positions and substitutions, employing a combinatorial optimization framework based on library-averaged energy potentials in order to avoid explicitly modeling every variant in every possible library. In case study applications to green fluorescent protein, b-lactamase, and lipase A, SOCoM optimizes relatively small, focused libraries whose variants achieve energies comparable to or better than previous library design efforts, as well as larger libraries (previously not designable by structure-based methods) whose variants cover greater diversity while still maintaining substantially better energies than would be achieved by representative random library approaches. By allowing the creation of large-scale combinatorial libraries based on structural calculations, SOCoM promises to increase the scope of applicability of computational protein design and improve the hit rate of discovering beneficial variants. While designs presented here focus on variant stability (predicted by total energy), SOCoM can readily incorporate other structure-based assessments, such as the energy gap between alternative conformational or bound states.

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“…Random multi-recombinant PCR (RM-PCR) (Tsuji et al, 2001) does not require homologous sequences at the recombination sites but the accessible number and size of the recombined fragments is very limited due to the reliance on synthetic DNA sequences. In sequence-independent sitedirected chimeragenesis (SISDC) (Hiraga and Arnold, 2003;Meyer et al, 2006a), type IIb restriction enzymes that cut outside their recognition site have been used, but their introduction is laborious and time consuming: the DNA fragments flanked by suitable restriction sites are first prepared by PCR, and then reassembled into their parent genes by overlap extension PCR. Moreover, only four fragments can be efficiently recombined at a time and intermediate cloning steps are required (Meyer et al, 2006a).…”

Section: Introductionmentioning

confidence: 99%

USER friendly DNA recombination (USERec): a simple and flexible near homology-independent method for gene library construction

Villiers¹,

Stein²,

Hollfelder³

2009

Protein Engineering, Design and Selection

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USER friendly DNA recombination (USERec) is introduced as a near homology-independent method that allows the simultaneous recombination of an unprecedented number of 10 DNA fragments (approximately 40-400 bp) within a day. The large number of fragments and their ease of preparation enables the creation of libraries of much larger genetic diversity (potentially approximately 10(10)-10(11) sequences) than current alternative methods based on DNA truncation (ITCHY, SCRATCHY and SHIPREC) or type IIb restriction enzymes (SISDC). At the same time, the frequency of frameshifts in the recombined library is low (90% of the recombined sequences are in frame). Compared to overlap extension PCR, USERec also requires much reduced crossover sequence constraints (only a 5'-AN(4-8)T-3' motif) and fewer experimental steps. Based on its simplicity and flexibility, and the accessibility of large and high quality recombined DNA libraries, USERec is established as a convenient alternative for the combinatorial assembly of gene fragments (e.g. exon or domain shuffling) and for a number of applications in gene library construction, such as loop grafting and multi-site-directed or random mutagenesis.

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Combinatorial Recombination of Gene Fragments to Construct a Library of Chimeras

Cited by 8 publications

References 25 publications

Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes

Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes

Structure‐based design of combinatorial mutagenesis libraries

USER friendly DNA recombination (USERec): a simple and flexible near homology-independent method for gene library construction

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