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Anticipatory evolution and DNA shuffling
Jamie M Bacher, Brian D Reiss, Andrew D Ellington
Genome Biology , 2002, DOI: 10.1186/gb-2002-3-8-reviews1021
Abstract: Proteins are machines created by evolution, but it is unclear just how finely evolution has guided their sequence, structure, and function. It is undoubtedly true that individual mutations in a protein affect both its structure and its function and that such mutations can be fixed during evolutionary history, but it is also true that there are other elements of protein sequence that have been acted upon by evolution. For example, the genetic code appears to be laid out so that mutations and errors in translation are minimally damaging to protein structure and function [1]. Could the probability that a beneficial mutation is found and fixed in the population also have been manipulated during the course of evolution, so that the proteins we see today are more capable of change than the proteins that may have been cobbled together following the 'invention' of translation? Have proteins, in fact, evolved to evolve? There is already some evidence that bacteria are equipped to evolve phenotypes that are more capable of further adaptation (reviewed in [2,3,4]). For example, mutator [5] and hyper-recombinogenic [6] strains arise as a result of selection experiments. The development of DNA shuffling (reviewed in [7,8]) and the appearance of several recent papers using this technique [9,10,11] provide us with a surprising new opportunity to ask and answer these fundamental questions at the level of individual genes, and perhaps even genomes.DNA shuffling, a method for in vitro recombination, was developed as a technique to generate mutant genes that would encode proteins with improved or unique functionality [12,13]. It consists of a three-step process that begins with the enzymatic digestion of genes, yielding smaller fragments of DNA. The small fragments are then allowed to randomly hybridize and are filled in to create longer fragments. Ultimately, any full-length, recombined genes that are recreated are amplified via the polymerase chain reaction. If a series of alleles o
Sulfolobus tokodaii RadA paralog, stRadC2, is involved in DNA recombination via interaction with RadA and Hjc
Lei Wang,DuoHong Sheng,WenYuan Han,Bin Huang,ShanShan Zhu,JinFeng Ni,Jia Li,YuLong Shen
Science China Life Sciences , 2012, DOI: 10.1007/s11427-012-4292-0
Abstract: Rad51/RadA paralogs found in eukaryotes and euryarchaea play important roles during recombination and repair, and mutations in one of the human Rad51 paralogs, Rad51C, are associated with breast and ovarian cancers. The hyperthermophilic crenarchaeon Sulfolobus tokodaii encodes four putative RadA paralogs and studies on these proteins may assist in understanding the functions of human Rad51 paralogs. Here, we report the biochemical characterization of stRadC2, a S. tokodaii RadA paralog. Pull-down assays revealed that the protein was able to interact with the recombinase, RadA, and the Holliday junction endonuclease, Hjc. stRadC2 inhibited the strand exchange activity of RadA and facilitated Hjc-mediated Holliday junction DNA cleavage in vitro. RT-PCR analysis revealed that stRadC2 transcription was immediately reduced after UV irradiation, but was restored to normal levels at the late stages of DNA repair. Our results suggest that stRadC2 may act as an antirecombination factor in DNA recombinational repair in S. tokodaii.
The sectionalized DNA shuffling: an effective tool for molecular directed evolution of Meiothermus ruber TreS
分段DNA shuffling: 一种大分子海藻糖合酶有效的定向进化方法

Liu Yan-Chao,Wang Yu-Fan,Qian Ke-Fan,Zhang Jun,Xiao Chen-Peng,Xing Lai-Jun,Li Ming-Chun,

微生物学通报 , 2013,
Abstract: Objective] The gene M-treS from Meiothermus ruber CBS-01 encodes a trehalose synthase of 962 amino acids, named M-TreS. To improve its catalytic activity, we constructed a method of molecular directed evolution, the sectionalized DNA shuffling. Methods] Through two PCR steps with two pairs of partially complementary primers, the M-treS gene was parted into two sections. After the two sections shuffled respectively, a whole gene was obtained through the complementarity of the primers. This method was more feasible, with higher mutability than normal DNA shuffling. Results] Mutants were obtained after one round of the sectionalized DNA shuffling, in combination with error-prone PCR. The best mutant enzyme contained 6 amino acid substitutions, whose catalytic activity and efficiency were 1.6-fold and 2-fold of that of the wild type, respectively. In the 6 amino acid substitutions, 5 were caused by homologous recombination, and one by error-prone PCR. Conclusion] This study indicates that the sectionalized DNA shuffling is an effective tool for molecular directed evolution of macromolecular proteins.
Partial exchangeability of the prior via shuffling  [PDF]
Erik van Zwet
Statistics , 2014,
Abstract: In inference problems involving a multi-dimensional parameter $\theta$, it is often natural to consider decision rules that have a risk which is invariant under some group $G$ of permutations of $\theta$. We show that this implies that the Bayes risk of the rule is {\em as if} the prior distribution of the parameter is partially exchangeable with respect to $G$. We provide a symmetrization technique for incorporating partial exchangeability of $\theta$ into a statistical model, without assuming any other prior information. We refer to this technique as {\em shuffling}. Shuffling can be viewed as an instance of empirical Bayes, where we estimate the (unordered) multiset of parameter values $\{\theta_1,\theta_2,\dots,\theta_p\}$ while using a uniform prior on $G$ for their ordering. Estimation of the multiset is a missing data problem which can be tackled with a stochastic EM algorithm. We show that in the special case of estimating the mean-value parameter in a regular exponential family model, shuffling leads to an estimator that is a weighted average of permuted versions of the usual maximum likelihood estimator. This is a novel form of shrinkage.
A novel method of DNA shuffling without PCR process
Qiang Wang,Qiuyun Liu,Gang Li,Baojian Li
Chinese Science Bulletin , 2004, DOI: 10.1007/BF03184266
Abstract: Most DNA shuffling methods currently used require PCR process. A novel method of DNA shuffling without PCR process is described, taking advantage of the feature of some restriction enzymes whose recognition sites differ from their cleavage sites, thus giving rise to different cohesive ends. These cohesive ends can be rejoined at their native sites from different parental sequences, generating new sequences with various combinations of mutations.
A novel method of DNA shuffling without PCR process
WANG Qiang,LIU Qiuyun,LI Gang & LI Baojian Key Laboratory of Gene Engineering of Ministry of Education,School of Life Sciences,Zhongshan University,Guangzhou,China Correspondence should be addressed to Li Baojian,

科学通报(英文版) , 2004,
Abstract: Most DNA shuffling methods currently used require PCR process. A novel method of DNA shuffling without PCR process is described, taking advantage of the feature of some restriction enzymes whose recognition sites differ from their cleavage sites, thus giving rise to different cohesive ends.These cohesive ends can be rejoined at their native sites from different parental sequences, generating new sequences with various combinations of mutations.
Which way up? Recognition of homologous DNA segments in parallel and antiparallel alignment  [PDF]
Dominic J. Lee,Aaron Wynveen,Tim Albrecht,Alexei A. Kornyshev
Quantitative Biology , 2014,
Abstract: Homologous gene shuffling between DNA promotes genetic diversity and is an important pathway for DNA repair. For this to occur, homologous genes need to find and recognize each other. However, despite its central role in homologous recombination, the mechanism of homology recognition is still an unsolved puzzle. While specific proteins are known to play a role at later stages of recombination, an initial coarse grained recognition step has been proposed. This relies on the sequence dependence of the DNA structural parameters, such as twist and rise, mediated by intermolecular interactions, in particular electrostatic ones. In this proposed mechanism, sequences having the same base pair text, or are homologous, have lower interaction energy than those sequences with uncorrelated base pair texts; the difference termed the recognition energy. Here, we probe how the recognition energy changes when one DNA fragment slides past another, and consider, for the first time, homologous sequences in antiparallel alignment. This dependence on sliding was termed the recognition well. We find that there is recognition well for anti-parallel, homologous DNA tracts, but only a very shallow one, so that their interaction will differ little from the interaction between two nonhomologous tracts. This fact may be utilized in single molecule experiments specially targeted to test the theory. As well as this, we test previous theoretical approximations in calculating the recognition well for parallel molecules against MC simulations, and consider more rigorously the optimization of the orientations of the fragments about their long axes. The more rigorous treatment affects the recognition energy a little, when the molecules are considered rigid. However when torsional flexibility of the DNA molecules is introduced, we find excellent agreement between analytical approximation and simulation.
Revealing biases inherent in recombination protocols
Javier F Chaparro-Riggers, Bernard LW Loo, Karen M Polizzi, Phillip R Gibbs, Xiao-Song Tang, Mark J Nelson, Andreas S Bommarius
BMC Biotechnology , 2007, DOI: 10.1186/1472-6750-7-77
Abstract: We developed different test systems to compare and reveal biases from DNA shuffling and recombination-dependent PCR (RD-PCR), a StEP-like recombination protocol. An assay based on the reactivation of β-lactamase was developed to simulate the recombination of point mutations. Both protocols performed similarly here, with slight advantages for RD-PCR. However, clear differences in the performance of the recombination protocols were observed when applied to homologous genes of varying DNA identities. Most importantly, the recombination-dependent PCR showed a less pronounced bias of the crossovers in regions with high sequence identity. We discovered that template variations, including engineered terminal truncations, have significant influence on the position of the crossovers in the recombination-dependent PCR. In comparison, DNA shuffling can produce higher crossover numbers, while the recombination-dependent PCR frequently results in one crossover. Lastly, DNA shuffling and recombination-dependent PCR both produce counter-productive variants such as parental sequences and have chimeras that are over-represented in a library, respectively. Lastly, only RD-PCR yielded chimeras in the low homology situation of GFP/mRFP (45% DNA identity level).By comparing different recombination scenarios, this study expands on existing recombination knowledge and sheds new light on known biases, which should improve library-creation efforts. It could be shown that the recombination-dependent PCR is an easy to perform alternative to DNA shuffling.Directed evolution of proteins has become a widely adopted and accepted method for protein engineering. There are two basic iterative steps involved in the process: the creation of diversity at the gene level and the screening or selection for improved variants [reviewed in [1-3]]. The quality of the diversity method is crucial and the performance of the chosen protocol has a direct impact on the success rate of obtaining improved variants as
DNA shuffling of Arabidopsis thalianna K+ uptake transporter gene

GUO Zhao-kui,YANG Qian,YAO Quan-hong,WAN Xiu-qing,YAN Pei-qiang,

中国生物工程杂志 , 2006,
Abstract: The DNA fragment sized 2139bp which sequence is the same with AtKup1 gene from Arabidopsis thalianna was used as the templates to do DNA family shuffling. The shuffled AtKup1 gene library was expressed in the mutant of S. cerevisae in which potassium transporter gene TRK1 and TRK2 were knocked out by homologous recombination. Then the screening was carried out in the low potassium media containing 5.0 mM KCl and no histidine in it. it was found that both of diverse and wild AtKup1 genes can rescue the trk1 trk2 yeast mutant strain on low K+] medium. The growth of 2 clones yeast contained diverse AtKup1 were beter than that of AtKup1 wild gene transformant. The sequencing results of the shuffled Atkup1 showed that there were 2 nucleotides had changed and resulted in 2 amino acid variations in it compared with original AtKup1. The potassium uptaking capacity of shuffled Atkup1 gene increased significantly when it was transformed into tobacco.
Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes  [PDF]
Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0005553
Abstract: 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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