Uniform amplification of phage with different growth characteristics in individual compartments consisting of monodisperse droplets.
Uniform amplification of phage with different growth characteristics in individual compartments consisting of monodisperse droplets.
复制标题
DOI：
10.1002/anie.201001143
复制
发表时间：
2010-07-19
影响因子：
16.6
通讯作者：
Whitesides, George M.
中科院分区：
文献类型：
--
作者：
Derda, Ratmir;Tang, Sindy K. Y.;Whitesides, George M.
噬菌体克隆混合物的均匀扩增对于噬菌体外壳蛋白上呈递的肽和蛋白质的选择（噬菌体展示）是关键的。[1，2]当具有不同生长速率的噬菌体在共同溶液中彼此竞争时，不能实现均匀扩增。在这里，我们描述了一种方法，从具有不同生长特性的克隆混合物中均匀扩增单个噬菌体克隆。我们使用微流控液滴发生器[3]将单个克隆从缓慢生长（S）和快速生长（R）的M13丝状噬菌体的混合物中分离到生长培养基的液滴中（ca.直径200 μm）。杆菌在足够低的噬菌体浓度下，每个液滴含有一个或不含有噬菌体颗粒。不同的噬菌体在分离于不同的液滴中时不能竞争细菌宿主，并且在扩增后保留了起始时存在的S和R克隆的相对数量。因为噬菌体克隆的扩增取决于它们所驻留的液滴的大小，所以使用均匀大小的液滴对于该过程的成功是必不可少的。在噬菌体的外壳蛋白上展示随机肽序列使得有可能产生具有很大多样性（> 109）的肽的文库;多轮选择和扩增使得有可能选择与许多靶有效结合的肽。[4-6]然而，噬菌体外壳蛋白的修饰可以影响细菌的感染速率、新噬菌体颗粒的组装速率或它们从被感染的细菌产生的速率。[1，2]当具有不同外壳蛋白组成的噬菌体（因此具有不同的扩增速率）竞争相同的细菌库时，复制更快的克隆捕获总细菌库的增加部分。噬菌体文库的扩增导致选择比文库的其余部分扩增更快的克隆;[7-9]它降低了文库的多样性并导致潜在有价值克隆的损失。例如，一个109个不同克隆的文库可以包含102-103个克隆，这些克隆展示的肽对靶标具有相似的亲和力;[10]但是几轮的选择和扩增可能只保留少数与靶标结合并快速扩增的克隆。当靶具有许多潜在的结合位点（例如细胞、器官）时，基于扩增的选择的问题加剧。[8，11，12]在扩增过程中消除不同噬菌体克隆之间的不期望的竞争将使得能够独立于它们的相对复制速率选择更宽的靶结合噬菌体库。我们演示了一个完成这种消除的过程，我们的演示使用了非裂解性的M13丝状噬菌体，这个噬菌体使用了E。coli作为宿主，并产生ca. 1000噬菌斑形成单位（pfu）的噬菌体在细菌感染的一小时内。[2]为了模拟噬菌体克隆之间的竞争，我们使用了两组克隆：1）经工程改造以在PIII外壳蛋白上呈递12-mer肽的M13噬菌体的市售文库，和2）野生型（wt）噬菌体（也称为“环境噬菌体”）。因为wt比工程化的噬菌体更有效地感染细菌，所以wt是“快速生长的噬菌体”（R）的极好模型，而工程化的文库提供了“缓慢生长的噬菌体”（S）的模型。在我们的实验中，S噬菌体（排他地）含有半乳糖苷酶报告基因，并在含有比色半乳糖苷酶底物X-gal的固体培养基上的细菌菌落中形成蓝色噬斑。[13]无报告基因的R噬菌体在相同条件下形成透明噬斑。
Uniform amplification of a mixture of phage clones is central to the selection of peptides and proteins presented on the coat proteins of phage (phage display).[1, 2] Uniform amplification cannot be achieved when phage having different rates of growth compete with each other in a common solution. Here we describe a method for uniform amplification of individual phage clones, from a mixture of clones possessing different growth characteristics. We use a microfluidic droplet generator [3] to separate individual clones from a mixture of slowly growing (S) and rapidly growing (R) M13 filamentous phage into droplets of growth media (ca. 200 μm in diameter) containing E. coli. At sufficiently low concentrations of phage, each droplet contains one or no phage particles. Different phage cannot compete for bacterial hosts when isolated in different droplets, and the relative number of S and R clones present at the start is preserved after amplification. Because amplification of phage clones depends on the size of the droplets in which they reside, the use of droplets of uniform size is essential for the success of this process. Display of random peptide sequences on the coat proteins of bacteriophages makes it possible to generate libraries of peptides of great diversity (> 109); rounds of selection and amplification make it possible to select peptides that bind usefully to many targets.[4–6] Modifications of phage coat proteins, however, can influence the rates of infection of bacteria, the rates of assembly of the new phage particles, or the rates of their production from infected bacteria.[1, 2] When phage with different compositions of the coat proteins (and thus different rates of amplification) compete for the same pool of bacteria, clones that replicate more rapidly capture an increasing fraction of the total pool of bacteria. Amplification of libraries of phage leads to selection of the clones that amplify faster than the rest of the library;[7–9] it decreases the diversity of the library and leads to loss of potentially valuable clones. For example, a library of 109 diverse clones can contain 102–103 of clones displaying peptides that have similar affinities for a target;[10] but rounds of selection and amplification are likely to retain only a few that bind to target and amplify rapidly. The problem of amplification-based selection is exacerbated when the target has many potential binding sites (eg cells, organs).[8, 11, 12] Elimination of undesired competition between different phage clones during amplification would enable selection of wider repertoire of targetbinding phage independently of their relative rates of replication. We demonstrate a procedure that accomplishes this elimination.Our demonstration used non-lytic M13 filamentous phage; this phage uses E. coli as a host, and produces a burst of ca. 1000 plaque-forming units (pfu) of phage within an hour of bacterial infection.[2] To model competition between phage clones, we used two sets of clones: 1) a commercially available library of M13 phage that was engineered to present a 12-mer peptide on the PIII coat protein, and 2) a wild-type (wt) phage (also known as “environmental phage”). Because wt infects bacteria more effectively than engineered phage, wt is an excellent model of a “rapidly growing phage”(R), while the engineered library provides a model of a “slowly growing phage”(S). In our experiments, S phage (exclusively) contained a galactosidase reporter, and formed blue plaques in bacterial colonies on solid media containing the colorimetric galactosidase substrate X-gal.[13] Reporter-free R phage forms clear plaques in the same conditions.
登录
查看更多内容
DOI：
10.1073/pnas.91.13.6093
发表时间：
1994-06-21
影响因子：
11.1
作者：
BREAKER, RR;JOYCE, GF
通讯作者：
JOYCE, GF
影响因子：
82.9
作者：
Arap, W;Kolonin, MG;Pasqualini, R
通讯作者：
Pasqualini, R
影响因子：
56.9
作者：
DEVLIN, JJ;PANGANIBAN, LC;DEVLIN, PE
通讯作者：
DEVLIN, PE
影响因子：
15
作者：
Derda, Ratmir;Musah, Samira;Orner, Brendan P.;Klim, Joseph R.;Li, Lingyin;Kiessling, Laura L.
通讯作者：
Kiessling, Laura L.
影响因子：
9.8
作者：
Halpin DR;Harbury PB
通讯作者：
Harbury PB