GeneArt® Synechococcus Protein Expression Kit - FAQs

pSyn_1的启动子是Psc。Psc是一种弱组成型启动子，可启动目的基因的基础表达。而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。

下列文章对叶绿体转化与核转化的优势做了一些讨论，请查看：

•Fischer et al. (2004) Plant-based production of biopharmaceuticals. Curr Opin Plant Biol 7:152–158.
•Kindle et al. (1991_ Engineering the chloroplast genome: Techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 88:1721–1725.

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。

聚球藻在保存6个月后不会丧失活力。我们尚未对超过6个月的保存时间进行测试。

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。

使用的是Synechococcus elongatus PCC 7942，一种模型蓝藻。

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）。

可通过OD750检测细胞生长，线性范围将在0.2-1.2（在1 cm光路中）之间。如果所得OD值过高，请稀释并再次检测。完全生长通常需要5-6天。可利用Life Technologies的Countess细胞计数仪或Tali细胞计数仪进行精确检测。计算细胞数量的公式如下：

细胞浓度（细胞/mL）= (OD750 – 0.088)/9 × 108 = (OD750 – 0.088)/(9 × 10-8)

请注意，我们的COA指出，“将240 μl的冻存细胞复苏，并使用6 mL Gibco TAP培养基在28°C和50 μE下培养。生长6天后，在750 nm下检测光密度。光密度必须达到0.6。每批次至少取6管进行存活率测试”。

GeneArt基因合成被用于合成质粒骨架。同时，GeneArt是合成生物学的伞型品牌，产品覆盖面非常广，也包含了这些藻类试剂盒。

可能存在。很遗憾，基因沉默是藻类表达中的一个大问题。沉默程度取决于基因序列和从该基因表达到细胞中的蛋白的毒性。

基因工程试剂盒专用于随机插入目的基因，这会导致目的基因快速丢失。该蛋白表达试剂盒含有pChlamy_4载体，经过设计可表达出带有博来霉素/Zeocin抗性基因sh-ble的转录融合蛋白（Rasala et al., 2012）。口蹄疫病毒（FMDV）2A肽段的自切割序列位于抗生素抗性基因和目的基因之间。该序列可编码一个约20个氨基酸的短序列，介导发生适当切割并产生2个独立蛋白。我们使用该系统得到的阳性转化株，即使在有或无筛选压力下传代多次之后，依然能够维持高表达水平，其持续时间远远长于其他系统。

是稳定转染。

平均预期时间常数是15-20毫秒。我们的使用手册中有Neon电穿孔法实验方案。

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 for pSyn_6, on the other hand, is the psbA promoter (PpsbA). PpsbA is a strong constitutive promoter of the psbA gene (encoding photosystem II protein D1) from Synechococcus elongatus, driving the high level expression of your gene of interest.

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

Please view the following articles that have some discussion on the advantages of chloroplast transformation versus nuclear transformation:

- Fischer et al. (2004) Plant-based production of biopharmaceuticals. Curr Opin Plant Biol 7:152-158.
- Kindle et al. (1991) Engineering the chloroplast genome: Techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 88:1721-1725.

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

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.

After 6 months of storage, the Synechococcus did not lose their competency. We have not yet tested longer storage points.

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

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.

We no longer offer any strains. Our reagents and protocols were developed using Synechococcus elongatus strain PCC 7942, a model cyanobacterium.

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

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.

The cell growth can be measured by OD750 and the linear range will be between 0.2 and 1.2 (in 1 cm lightpass). If too high, please dilute them and measure again. It usually takes 5-6 days for full growth. An accurate measure can be taken using the Countess Cell Counter or Tali Cytometer from Thermo Fisher Scientific. The formula to calculate cell number is as follows:

Cell concentration (cells/mL) = (OD750 - 0.088)/9 × 10e8 = (OD750 - 0.088)/(9 × 10-8)

Please note, our COA states that “240 µl of frozen cells are thawed out and cultured in 6 mL of Gibco TAP media at 28 degrees C and 50 µE light. Optical density is measured at 750 nm following 6 days of growth. Optical density must reach 0.6. Viability testing is performed on a minimum of 6 vials per lot.”

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

GeneArt gene synthesis was used to create the plasmid backbones synthetically. Also, GeneArt is the umbrella brand for synthetic biology, which is a large area in which these kits will be used.

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

Yes, unfortunately, silencing is a big problem in algae. The extent of silencing depends on the sequence of the gene and the toxicity of the protein that expresses from that gene to the cell.
The engineering kit is designed for random insertion of your gene of interest, which can lead to your gene being lost quickly. The protein expression kit, which contains the pChlamy_4 vector, is designed so that proteins are expressed as transcriptional fusions with the bleomycin/zeocin resistance gene sh-ble (Rasala et al., 2012). The self-cleaving sequence for the 2A peptide from the foot-and-mouth disease virus (FMDV) is placed between the antibiotic resistance gene and the gene of interest. It encodes a short ~20 amino acid sequence that mediates proper cleavage to yield two discrete proteins. With this system we have seen positive transformants maintain high expression levels for much longer than with other systems, even after many passages with or without selection pressure.

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

The average expected time constant is 15-20 milliseconds. We have a Neon electroporation protocol in the manual.

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