New Vectors for Chromosomal Integration Enable High-Level Constitutive or Inducible Magnetosome Expression of Fusion Proteins in Magnetospirillum gryphiswaldense
New Vectors for Chromosomal Integration Enable High-Level Constitutive or Inducible Magnetosome Expression of Fusion Proteins in Magnetospirillum gryphiswaldense
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DOI：
10.1128/aem.00192-14
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发表时间：
2014-04-01
影响因子：
4.4
通讯作者：
Schueler, Dirk
中科院分区：
文献类型：
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作者：
Borg, Sarah;Hofmann, Julia;Schueler, Dirk
甲蛋白细菌能使磁小体发生生物矿化，磁小体由单晶磁铁矿核心组成，包裹着一层含有特定蛋白质的磷脂双层。磁小体代表了具有前所未有的磁性和物理化学特性的磁性纳米颗粒。这使得它们在许多生物技术和生物医学应用中具有潜在的实用价值。通过与磁小体锚肽的基因融合，表达外源蛋白可以实现进一步的功能化。然而，可用于在趋磁细菌中强而可控的蛋白质表达的遗传工具集是非常有限的。在这里，我们描述了多用途的载体，无论是可诱导的或高水平的组件性表达的蛋白质在格雷菲斯瓦尔登斯。将基因工程的P-MAMDC启动子与密码子优化的EGFP基因(MAG-EGFP)相结合，使组成性表达增加8倍，并产生更明亮的荧光。我们进一步证明了广泛使用的P-tet启动子在灰指藻中具有功能性和可调性。通过Tn5介导的单拷贝染色体插入，实现了绿色荧光蛋白和β-葡萄糖醛酸酶(GUSA)报告基因的稳定和均匀表达。此外，MAG-EGFP-EGFP融合到MamC的基因复制导致磁小体表达和荧光进一步增强。据估计，每个磁小体颗粒的GFP拷贝数在80到210(对于单个MamC-Mag-EGFP)和200到520(对于MamC-Mag-EGFP-EGFP)之间。
The alphaproteobacterium Magnetospirillum gryphiswaldense biomineralizes magnetosomes, which consist of monocrystalline magnetite cores enveloped by a phospholipid bilayer containing specific proteins. Magnetosomes represent magnetic nanoparticles with unprecedented magnetic and physicochemical characteristics. These make them potentially useful in a number of biotechnological and biomedical applications. Further functionalization can be achieved by expression of foreign proteins via genetic fusion to magnetosome anchor peptides. However, the available genetic tool set for strong and controlled protein expression in magnetotactic bacteria is very limited. Here, we describe versatile vectors for either inducible or high-level constitutive expression of proteins in M. gryphiswaldense. The combination of an engineered native P-mamDC promoter with a codon-optimized egfp gene (Mag-egfp) resulted in an 8-fold increase in constitutive expression and in brighter fluorescence. We further demonstrate that the widely used P-tet promoter is functional and tunable in M. gryphiswaldense. Stable and uniform expression of the EGFP and beta-glucuronidase (GusA) reporters was achieved by single-copy chromosomal insertion via Tn5-mediated transposition. In addition, gene duplication by Mag-EGFP-EGFP fusions to MamC resulted in further increased magnetosome expression and fluorescence. Between 80 and 210 (for single MamC-Mag-EGFP) and 200 and 520 (for MamC-Mag-EGFP-EGFP) GFP copies were estimated to be expressed per individual magnetosome particle.