Phenotypic Expression of PCR-Generated Random Mutations in a Pseudomonas putida Gene after Its Introduction into an Acinetobacter Chromosome by Natural Transformation
Phenotypic Expression of PCR-Generated Random Mutations in a Pseudomonas putida Gene after Its Introduction into an Acinetobacter Chromosome by Natural Transformation
复制标题
通过自然转化导入不动杆菌染色体后恶臭假单胞菌基因中 PCR 产生的随机突变的表型表达
DOI：
--
复制
发表时间：
1999
影响因子：
4.4
通讯作者：
L. Ornston
中科院分区：
文献类型：
--
作者：
R. Kok;David M. Young;L. Ornston
摘要PCR扩增产生的随机点突变可以通过自然转化的方法有效地转移到不动杆菌ADP 1（也称为菌株BD 413）的染色体上。该技术不需要在质粒中克隆PCR片段：PCR扩增的DNA片段被细胞内化，并通过同源重组直接并入其基因组。以前，这种随机诱变的方法只能应用于不动杆菌基因，以提供突变体表型的选择。在这里，我们描述了一个载体和受体，允许诱变，回收和表达的异源基因，可能缺乏一个积极的选择。该质粒携带一个不动杆菌染色体片段，该片段被卡那霉素抗性标记旁的多克隆位点中断。将异源DNA插入多克隆位点制备插入物作为PCR诱变的靶标。PCR扩增卡那霉素抗性标记和不动杆菌DNA的侧翼区沿着异源DNA的插入。在工程化的不动杆菌受体中，侧翼区和相应的染色体区段之间的核苷酸序列同一性允许PCR扩增的DNA片段同源重组到特定的染色体对接位点中，它们可以从该位点表达。受体菌株仅含有卡那霉素抗性基因的一部分，因此可以通过要求重组体在卡那霉素存在下生长来选择含有该基因和诱变插入物的供体DNA。该技术的有效性证明了相对富含GC的恶臭假单胞菌xylE基因。在仅一轮PCR扩增（35个循环）后，供体DNA产生转化体，其中在37°C下生长后高达30%的转化体携带缺陷xylE基因。在37°C下不能表达xylE的重组克隆中，约10%在22°C下生长时表达该基因。本文所述的技术可适用于制备在任何基因中具有改变的功能的集落，对于所述基因存在选择或合适的表型筛选。
ABSTRACT Localized sets of random point mutations generated by PCR amplification can be transferred efficiently to the chromosome ofAcinetobacter ADP1 (also known as strain BD413) by natural transformation. The technique does not require cloning of PCR fragments in plasmids: PCR-amplified DNA fragments are internalized by cells and directly incorporated into their genomes by homologous recombination. Previously such procedures for random mutagenesis could be applied only to Acinetobacter genes affording the selection of mutant phenotypes. Here we describe the construction of a vector and recipient that allow for mutagenesis, recovery, and expression of heterologous genes that may lack a positive selection. The plasmid carries an Acinetobacterchromosomal segment interrupted by a multiple cloning site next to a kanamycin resistance marker. The insertion of heterologous DNA into the multiple cloning site prepares the insert as a target for PCR mutagenesis. PCR amplifies the kanamycin resistance marker and a flanking region of Acinetobacter DNA along with the insert of heterologous DNA. Nucleotide sequence identity between the flanking regions and corresponding chromosomal segments in an engineered Acinetobacter recipient allows homologous recombination of the PCR-amplified DNA fragments into a specific chromosomal docking site from which they can be expressed. The recipient strain contains only a portion of the kanamycin resistance gene, so donor DNA containing both this gene and the mutagenized insert can be selected by demanding growth of recombinants in the presence of kanamycin. The effectiveness of the technique was demonstrated with the relatively GC-rich Pseudomonas putida xylE gene. After only one round of PCR amplification (35 cycles), donor DNA produced transformants of which up to 30% carried a defective xylEgene after growth at 37°C. Of recombinant clones that failed to express xylE at 37°C, about 10% expressed the gene when grown at 22°C. The techniques described here could be adapted to prepare colonies with an altered function in any gene for which either a selection or a suitable phenotypic screen exists.
DOI：
10.1101/gr.2.1.28
发表时间：
1992-08-01
期刊：
PCR methods and applications
影响因子：
--
作者：
Cadwell, R C;Joyce, G F
通讯作者：
Joyce, G F