公开(公告)号：US20070269862A1

公开(公告)日：2007-11-22

申请号：US11644713

申请日：2006-12-22

申请人： John Glass ,  Lei Young ,  Carole Lartigue ,  Nacyra Assad-Garcia ,  Hamilton Smith ,  Clyde Hutchison ,  J. Venter

发明人： John Glass ,  Lei Young ,  Carole Lartigue ,  Nacyra Assad-Garcia ,  Hamilton Smith ,  Clyde Hutchison ,  J. Venter

IPC分类号： C12N15/87 ,  C12N5/06 ,  C12P21/04

CPC分类号： C12P21/02 ,  C12N2510/00

摘要： A method is provided for introducing a genome into a cell or cell-like system. The introduced genome may occur in nature, be manmade with or without automation, or may be a hybrid of naturally occurring and manmade materials. The genome is obtained outside of a cell with minimal damage. Materials such as a proteins, RNAs, polycations, nucleoid condensation proteins, or gene translation systems may accompany the genome. The genome is installed into a naturally occurring cell or into a manmade cell-like system. A cell-like system or synthetic cell resulting from the practice of the provided method may be designed and used to yield gene-expression products, such as desired proteins. By enabling the synthesis of cells or cell-like systems comprising a wide variety of genomes, accompanying materials and membrane types, the provided method makes possible a broader field of experimentation and bioengineering than has been available using prior art methods.

摘要翻译： 提供了将基因组导入细胞或细胞样系统的方法。 引入的基因组可以在自然界中发生，在有或没有自动化的情况下是人造的，或者可以是天然存在的和人造的材料的混合物。 基因组以最小的损伤在细胞外获得。 材料如蛋白质，RNAs，聚阳离子，核仁缩合蛋白或基因翻译系统可能伴随着基因组。 将基因组安装到天然存在的细胞或人造细胞样系统中。 可以设计并使用由所提供的方法的实践产生的细胞样系统或合成细胞来产生基因表达产物，例如所需的蛋白质。 通过实现包含各种各样的基因组，伴随材料和膜类型的细胞或细胞样系统的合成，所提供的方法可能比使用现有技术方法可用的更广泛的实验和生物工程领域。

公开(公告)号：US20070122826A1

公开(公告)日：2007-05-31

申请号：US11546364

申请日：2006-10-12

申请人： John Glass ,  Hamilton Smith ,  Clyde Hutchison ,  Nina Alperovich ,  Nacyra Assad-Garcia

发明人： John Glass ,  Hamilton Smith ,  Clyde Hutchison ,  Nina Alperovich ,  Nacyra Assad-Garcia

IPC分类号： C40B30/06 ,  C40B40/10 ,  C07H21/04 ,  C12P7/06 ,  C12P1/06 ,  C12N9/02 ,  C12N1/21

CPC分类号： C12N1/20 ,  C07K14/195 ,  C07K14/30 ,  C12N9/16 ,  C12P3/00 ,  C12P7/06 ,  C12R1/35 ,  C12Y301/04046 ,  Y02E50/17

摘要： The present invention relates, e.g., to a minimal set of protein-coding genes which provides the information required for replication of a free-living organism in a rich bacterial culture medium, wherein (1) the gene set does not comprise the 101 genes listed in Table 2; and/or wherein (2) the gene set comprises the 381 protein-coding genes listed in Table 3 and, optionally, one of more of: a set of three genes encoding ABC transporters for phosphate import (genes MG410, MG411 and MG412; or genes MG289, MG290 and MG291); the lipoprotein-encoding gene MG185 or MG260; and/or the glycerophosphoryl diester phosphodiesterase gene MG293 or MG385.

摘要翻译： 本发明涉及例如提供在富细菌培养基中复制自由活体的所需信息的蛋白质编码基因的最小集合，其中（1）基因组不包括所列出的101个基因 在表2中; 和/或其中（2）基因组包含表3中列出的381个蛋白质编码基因，以及任选的一个以下的一个：编码用于磷酸酯进口的ABC转运蛋白的三个基因（基因MG410，MG411和MG412;或 基因MG289，MG290和MG291）; 编码脂蛋白的基因MG185或MG260; 和/或甘油磷酸二酯磷酸二酯酶基因MG293或MG385。

公开(公告)号：US20070264688A1

公开(公告)日：2007-11-15

申请号：US11635355

申请日：2006-12-06

申请人： J. Venter ,  Hamilton Smith ,  Clyde Hutchison

发明人： J. Venter ,  Hamilton Smith ,  Clyde Hutchison

IPC分类号： C07H21/04 ,  C12N5/06 ,  C12P1/04

CPC分类号： C12N15/10 ,  C12N15/1093 ,  C12N15/66

摘要： Methods are provided for constructing a synthetic genome, comprising generating and assembling nucleic acid cassettes comprising portions of the genome, wherein at least one of the nucleic acid cassettes is constructed from nucleic acid components that have been chemically synthesized, or from copies of the chemically synthesized nucleic acid components. In one embodiment, the entire synthetic genome is constructed from nucleic acid components that have been chemically synthesized, or from copies of the chemically synthesized nucleic acid components. Rational methods may be used to design the synthetic genome (e.g., to establish a minimal genome and/or to optimize the function of genes within a genome, such as by mutating or rearranging the order of the genes). Synthetic genomes of the invention may be introduced into vesicles (e.g., bacterial cells from which part or all of the resident genome has been removed, or synthetic vesicles) to generate synthetic cells. Synthetic genomes or synthetic cells may be used for a variety of purposes, including the generation of synthetic fuels, such as hydrogen or ethanol.

摘要翻译： 提供了用于构建合成基因组的方法，包括产生和组装包含基因组部分的核酸盒，其中至少一个核酸盒由化学合成的核酸组分构成，或者由化学合成的 核酸成分。 在一个实施方案中，整个合成基因组由已化学合成的核酸组分或化学合成的核酸组分的拷贝构建。 合理的方法可用于设计合成基因组（例如，建立最小基因组和/或优化基因组内的基因的功能，例如通过突变或重新排列基因的顺序）。 可以将本发明的合成基因组引入囊泡（例如，其中已经除去部分或全部常驻基因组的细菌细胞或合成的囊泡）以产生合成细胞。 合成基因组或合成细胞可以用于各种目的，包括生成合成燃料，例如氢或乙醇。

公开(公告)号：US20050131222A1

公开(公告)日：2005-06-16

申请号：US10981687

申请日：2004-11-05

申请人： Robert Fleischmann ,  Mark Adams ,  Owen White ,  Hamilton Smith ,  J. Venter

发明人： Robert Fleischmann ,  Mark Adams ,  Owen White ,  Hamilton Smith ,  J. Venter

IPC分类号： C07K14/285 ,  C07H21/04

CPC分类号： C07K14/285

摘要： The present invention provides the sequencing of the entire genome of Haemophilus influenzae Rd, SEQ ID NO:1. The present invention further provides the sequence information stored on computer readable media, and computer-based systems and methods which facilitate its use. In addition to the entire genomic sequence, the present invention identifies over 1700 protein encoding fragments of the genome and identifies, by position relative to a unique Not I restriction endonuclease site, any regulatory elements which modulate the expression of the protein encoding fragments of the Haemophilus genome.

摘要翻译： 本发明提供了流感嗜血杆菌Rd，SEQ ID NO：1的整个基因组的测序。 本发明还提供存储在计算机可读介质上的序列信息，以及便于其使用的基于计算机的系统和方法。 除了整个基因组序列之外，本发明鉴定了基因组的超过1700个蛋白质编码片段，并且通过相对于独特的Not I限制性内切核酸酶位点的位置鉴定调节嗜血杆菌蛋白编码片段的表达的任何调节元件 基因组

公开(公告)号：US20070037197A1

公开(公告)日：2007-02-15

申请号：US11502746

申请日：2006-08-11

申请人： Lei Young ,  Hamilton Smith ,  Daniel Gibson

发明人： Lei Young ,  Hamilton Smith ,  Daniel Gibson

IPC分类号： C12Q1/68 ,  C12P19/34

CPC分类号： C12N15/902 ,  C12N9/1252 ,  C12N9/22 ,  C12N9/93 ,  C12N15/10 ,  C12N15/64 ,  C12N15/66 ,  C12P19/34 ,  C12Q1/62 ,  C12Q1/686 ,  C12Q1/6862 ,  C12Q2521/501 ,  C12Q2522/101 ,  C12Y207/07007 ,  C12Y301/11003 ,  C12Y605/01001

摘要： The present invention relates, e.g., to in vitro method, using isolated protein reagents, for joining two double stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising contacting the two DNA molecules in a reaction mixture with (a) a non-processive 5′ exonculease; (b) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing; (c) a non strand-displacing DNA polymerase; and (d) a ligase, under conditions effective to join the two DNA molecules to form an intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes.

摘要翻译： 本发明涉及例如使用分离的蛋白质试剂连接目标的两个双链（ds）DNA分子的体外方法，其中第一DNA分子的远端区域和第二DNA分子的近端区域共享一个 包括使反应混合物中的两个DNA分子与（a）非进程性5'外切酶接触; （b）加速核酸退火的单链DNA结合蛋白（SSB）; （c）非链置换DNA聚合酶; 和（d）连接酶，在有效连接两个DNA分子以形成完整双链DNA分子的条件下，其中保留序列同一性区域的单拷贝。 该方法允许以预定的顺序和取向连接多个DNA片段，而不使用限制酶。

公开(公告)号：US20070037196A1

公开(公告)日：2007-02-15

申请号：US11502624

申请日：2006-08-11

申请人： Daniel Gibson ,  Hamilton Smith

发明人： Daniel Gibson ,  Hamilton Smith

IPC分类号： C12Q1/68 ,  C12P19/34

CPC分类号： C12N9/1252 ,  C12N15/10 ,  C12N15/102 ,  C12N15/1031 ,  C12N15/64 ,  C12N15/66 ,  C12P19/34

摘要： The present invention relates, e.g., to an in vitro method, using isolated protein reagents, for joining two double-stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising (a) chewing back the DNA molecules with an enzyme having an exonuclease activity, to yield single-stranded overhanging portions of each DNA molecule which contain a sufficient length of the region of sequence identity to hybridize specifically to each other; (b) specifically annealing the single-stranded overhangs; and (c) repairing single-stranded gaps in the annealed DNA molecules and sealing the nicks thus formed (ligating the nicked DNA molecules). The region of sequence identity generally comprises at least 20 non-palindromic nucleotides (nt), e.g., at least about 40 non-palindromic nt. In some embodiments of the invention, about 5% PEG is present during all steps of the reaction, and/or the repair reaction is achieved with Taq DNA polymerase and a compatible ligase, such as Taq DNA ligase. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest.

摘要翻译： 本发明涉及例如使用分离的蛋白质试剂连接两个目的双链（ds）DNA分子的体外方法，其中第一DNA分子的远端区域和第二DNA分子的近端区域 共享序列同一性区域，其包括（a）用具有核酸外切酶活性的酶咀嚼DNA分子，以产生每个DNA分子的单链突出部分，其含有足够长度的序列同一性区域以与 彼此; （b）具体退火单链突出端; 和（c）修复退火的DNA分子中的单链间隙并密封由此形成的切口（连接有缺口的DNA分子）。 序列同一性区域通常包含至少20个非回文序列核苷酸（nt），例如至少约40个非回文性核苷酸。 在本发明的一些实施方案中，在反应的所有步骤期间存在约5％的PEG，和/或用Taq DNA聚合酶和相容连接酶如Taq DNA连接酶实现修复反应。 该方法允许以预定的顺序和取向连接多个DNA片段，而不使用限制酶。 其可以用于例如合成产生的感兴趣的基因或基因组的亚片段。