GeneArt™ Chlamydomonas Protein Expression Vector - FAQs

pChlamy_4载体专为避免发生常出现于C. reinhardtii的转基因沉默而设计。同时，该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。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。

需要。对于TOPO载体，您可通过设计引物，使编码序列与ATG一起位于载体的读码框内。

预期时间常数是15-20毫秒（平均为17毫秒）。

我们未报导衣藻细胞的转化效率，因为转化的最终结果是随机整合到基因组。电穿孔结果取决于目的基因。每个电穿孔反应中，对照载体应产生最少30个转录株。在每个挑选出的菌落中，目的基因阳性克隆数应不少于90%。

整合是随机的。基因工程试剂盒是基于随机插入，这会导致目的基因快速丢失。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。

很遗憾，我们尚未对其他藻类进行测试。

该试剂盒不是为长期保存阳性筛选克隆而设计的。大多数人在使用时将培养皿放在室温（不是4°C，因为它们需要光照）下的实验台上，根据需要反复划线。使用藻类的GeneArt低温保存试剂盒，可将藻株和克隆保存在–80°C数年。

Neon转染系统可用于电转衣藻。请参见GeneArt Chlamydomonas Protein Expression Vector (货号A24231)的说明书中推荐的电转方案。目前，我们尚未提供细菌电转的Neon实验方案。

The pChlamy_4 vector was designed in order to circumvent the transgene silencing that often occurs in C. reinhardtii. This vector is also designed so that proteins are expressed as transcriptional fusions with the blemoycin/zeocin resistance gene (sh-ble). 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.

Yes. For the TOPO version, you can design the primer to make sure the coding sequence is in frame with the ATG in the vector.

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

The expected time constant is 15-20 milliseconds (average is 17 milliseconds).

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

We don't report transformation efficiency of the Chlamydomonas cells, since the end result of transformation is random integration into the genome. The electroporation results will depend on the gene of interest. The control vector should produce a minimum of 30 transformants per electroporation reaction. Approximately 90% of colonies picked should be positive clones containing your gene of interest.

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

Integration is at random. The engineering kit is based on a random insertion in which the gene of interest can be lost very quickly. For the protein expression kit, the pChlamy_4 vector is designed so 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.

Unfortunately, we have not yet tested other algae.

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

The kits are not designed for long-term storage of positive-selected clones. Most people keep the plates on their bench top at room temperature (not 4 degrees C, as they need light) while in use, re-streaking if necessary. The GeneArt Cryopreservation Kit for Algae can be used to preserve algal strains and clones for storage at -80 degrees C for years.

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

The Neon Transfection System may be used for electroporation of Chlamydomonas. Please refer to the GeneArt Chlamydomonas Protein Expression Vector (Cat. No. A24231) manual for Neon instructions. Currently, we do not offer a Neon protocol for electroporation of bacterial cells.

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

The MAX Efficiency Transformation Reagent for Algae was developed for transforming the pChlamy_4 vector into the Chlamydomonas reinhardtii 137c cells by electroporation.

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

The GeneArt Cryopreservation Kit for Algae was developed for preserving the Chlamydomonas reinhardtii 137c cells. We have reports from some customers who were successfully able to preserve other algae cells.

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

It includes the pChlamy_4 vector. This vector has been tested for protein expression in Chlamydomonas reinhardtii 137c . You can get the Chlamydomonas reinhardtii 137c cells from the Chlamydomonas Resource Center (http://www.chlamycollection.org/).

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