Alternative Genetic Selection in Bacillus anthracis and Francisella tularensis

炭疽芽孢杆菌和土拉弗朗西斯菌的替代遗传选择

• 批准号: 7286467
• 负责人: GEORGE C. STEWART
• 金额: $ 18.69万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-05 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7286467
• 关键词: Antibiotic Resistance; Antibiotics; Arsenites; Bacillus anthracis; Bacteria; Cadmium; Categories; Characteristics; Chromates; Chromosomes; Condition; Engineering; Excision; Facility Construction Funding Category; Francisella tularensis; Genes; Genetic Markers; Goals; Heavy Metals; Herbicides; Mediating; Mercury; Mutate; Numbers; Operon; Organism; Phenotype; Plasmid Cloning Vector; Plasmids; Property; Research Project Grants; Resistance; Site; Streptomyces; System; antimicrobial; base; bialaphos; genetic analysis; genetic selection; mutant; recombinase

DESCRIPTION (provided by applicant): Many of the category A select agent bacteria are naturally sensitive to antibiotics and thus introduction of antibiotic resistance selective markers for genetic studies should be avoided. Non-antibiotic selective markers are limited in their availability and have not been evaluated for use in these bacteria. Markers to be developed should have the characteristics of having the selective phenotype expressed when the gene is present in single copy in the organism, being stable when expressed on multi-copy plasmids, and not exerting polar effects on downstream genes when inserted into a multi-gene operon. Desirable properties also include that the selective agent employed should give a low background when sensitive organisms are plated under selective conditions, and the difference between the MIC concentration of sensitive versus resistant organisms should be large. Furthermore, it would be advantageous to be able to easily and precisely excise the selective marker to create a mutant strain lacking the marker cassette so that the antimicrobial markers can be used iteratively to create multiply mutated strains. In this project, we will evaluate heavy metal resistance markers, including resistance to arsenite, cadmium, chromate, and mercury as well as resistance to the herbicide bialaphos as potential selective markers for genetic analysis of Bacillus anthracis and Francisella tularensis. The minimal sequence required for selection in the two hosts will be determined and mutational studies will be carried out to optimize expression. The use of these markers on multicopy plasmids and single copy insertions will be validated in the two hosts. Construction of a transposon bearing these selective agents will be created and evaluated. Engineering of the selective marker cassettes flanked by loxP sites will be accomplished and use of a Cre recombinase-based system to excise the selection cassette from plasmid or chromosomal targets in these select agent bacteria will be evaluated.

描述（由申请人提供）：许多A类选择剂细菌对抗生素天然敏感，因此应避免引入用于遗传研究的抗生素抗性选择标记。非抗生素选择性标记的可用性有限，并且尚未评估其在这些细菌中的使用情况。待开发的标记应具有以下特征：当基因在生物体中以单拷贝存在时具有选择性表型表达；在多拷贝质粒上表达时稳定；插入多拷贝质粒时不会对下游基因产生极性影响。基因操纵子。理想的特性还包括，当敏感生物体在选择性条件下铺板时，所使用的选择剂应该给出低背景，并且敏感生物体与抗性生物体的MIC浓度之间的差异应该很大。此外，能够容易且精确地切除选择性标记以产生缺乏标记盒的突变菌株将是有利的，使得抗微生物标记可以反复使用以产生多重突变菌株。在该项目中，我们将评估重金属抗性标记，包括对亚砷酸盐、镉、铬酸盐和汞的抗性以及对除草剂双丙磷的抗性，作为炭疽芽孢杆菌和土拉弗朗西斯菌遗传分析的潜在选择标记。将确定在两个宿主中选择所需的最小序列，并进行突变研究以优化表达。这些标记在多拷贝质粒和单拷贝插入上的使用将在两个宿主中得到验证。将创建并评估带有这些选择剂的转座子的构建。将完成两侧为 loxP 位点的选择性标记盒的工程设计，并将评估使用基于 Cre 重组酶的系统从这些选择剂细菌中的质粒或染色体靶标中切除选择盒的情况。