GeneArt™ Synechococcus Protein Expression Vector - FAQs

查看更多产品信息 GeneArt™ Synechococcus Protein Expression Vector - FAQs (A24230)

7 个常见问题解答

pSyn_1与pSyn_6有何区别？

pSyn_1的启动子是Psc。Psc是一种弱组成型启动子，可启动目的基因的本底表达。该启动子来自集胞藻PCC6803的茄呢酰二磷酸酯合酶基因。注意：该弱组成型启动子适用于受强表达阻碍的应用，如通路基因工程或低水平表达型突变基因的互补作用（Simkovsky et al., 2012）。

pSyn_6的启动子是psbA启动子（PpsbA）。PpsbA是细长聚球藻psbA基因（编码光合系统II蛋白D1）的一种强组成型启动子，可启动目的基因的高水平表达。pSyn-6载体还包括一个用于有效起始翻译的核糖体结合位点（GAAGGAG）和一个终止密码子。载体骨架还包含一个6xHis TEV和一个V5-His附加表位，用于检测/纯化目的基因。两种载体都含有NS1（中性位点1）同源重组位点，用于将载体整合到细长聚球藻基因组中。

使用聚球藻表达试剂盒进行整合的原理是什么？

在基因组中性位点1（Neutral Site 1）之间和质粒的NSa和NSb之间发生双重同源重组事件，从而将目的基因和壮观美素抗性整合到中性位点1内的基因组中。

我想通过非随机整合建立稳定的藻类。你们推荐使用哪种试剂盒？

推荐使用我们的Synechococcus试剂盒，其整合是针对藻类基因组的中性位点1（Neutral Site 1）。

What is the difference between pSyn_1 and pSyn_6?

The promoter for pSyn_1 is the Psc. Psc is a weak constitutive promoter that drives basal expression of your gene of interest. The promoter was taken from the solanesyl diphosphate synthase gene from Synechocystis sp. Strain PCC 6803. Note: Use of this weak constitutive promoter is a good fit for applications that are hindered by strong expression, such as pathway engineering or complementation of mutant genes normally expressed at low levels (Simkovsky et al., 2012).

The promoter for pSyn_6 is the psbA promoter (PpsbA). PpsbA is a strong constitutive promoter of the psbA gene (encoding photosystem II protein D1) from Synechococcus elongatus, driving high-level expression of your gene of interest. The pSyn-6 vector also includes a ribosome-binding site (GAAGGAG) for efficient initiation of translation, as well as a stop codon. A 6xHis TEV and a V5-His epitope tag is also included in the vector backbone for detection/purification of the gene of interest. NS1 (neutral site 1) homologous recombination sites are present in both vectors for the integration of the vector into the Synechococcus elongatus genome.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What is the mechanism of integration when using the Synechococcus expression kits?

A double homologous recombination event occurs between the Neutral Site 1 in the genome and between NSa and NSb in the plasmid. This results in the gene of interest and spectinomycin resistance integrating into the genome within the Neutral Site 1.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I would like to create a stable algae line via non-random integration. Which kit would you recommend?

We recommend our Synechococcus kit (A24230), where the integration is directed to the Neutral Site 1 of the algae genome.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What does the GeneArt Synechococcus Protein Expression Vector include?

It includes the pSyn_6 vector. This vector has been tested for protein expression in the Synechococcus elongatus PPC 7942. You can get the Synechococcus elongatus PPC 7942 from academic resources (https://genome.jgi.doe.gov/portal/synel/synel.info.html) or from the ATCC Collection (https://www.atcc.org/products/all/33912.aspx).

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.