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查看更多产品信息 GeneArt® Chlamydomonas Protein Expression Kit - FAQs (A24244)
49 个常见问题解答
请查看以下建议：
1. 为得到最佳结果，藻类细胞浓度应在1 x 106至2 x 106细胞/mL之间（OD范围为0.3-0.5）。细胞浓度不可超过3 x 106细胞/mL。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OD值超出线性范围，请稀释并再次检测以获得准确读数。
公式为：细胞数= (OD750 – 0.088)/ 9 × 106 /mL
2. 线性DNA转化比环状DNA转化的效率高很多（有效率约高出70%）。
3. DNA的质量和浓度对于转化效率至关重要。您将需要使用PureLink HQ、PureLink HiPure或相同质量的质粒纯化试剂盒进行DNA纯化。使用小体积的浓缩纯化DNA要比使用大体积的低浓度DNA更好。（如果您在转化线性质粒，则在线性化之后，您将需要在转化前使用凝胶提取或PCR净化试剂盒对质粒进行纯化处理。）我们建议每次电穿孔使用2 µg的线性质粒DNA。
4. 质粒DNA是随机插入到基因组的。通常，只有20%的转化株会明显表达目的基因。我们建议首先通过菌落PCR对菌落进行筛选，确保启动子和目的基因的完全整合，随后通过筛选多个阳性克隆，挑选出表达水平最高的克隆。
5. 由于C. reinhardtii基因组具有非常高的GC含量（约62% GC），如果目的基因适应了高表达C. reinhardtii基因的密码子使用偏好，则重组基因的表达水平会明显改善。
6. 由于转化效率取决于构建体向基因组的随机整合，电穿孔结果将取决于目的基因的性质。您可尝试转化试剂盒中的对照载体，从而确认试剂盒及其电穿孔方法是有效的。
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。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。
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.
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。
pChlamy_1载体无终止密码子和3' UTR。pChlamy_3载体含有3’UTR，已被证明可增强蛋白表达。
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。
是的，pChlamy_3载体在多克隆位点后含有一个3’ UTR。
建议，如果您想使用pChlamy_1载体表达高水平的重组蛋白，您的插入片段需要在紧随终止密码子后的位置含有一个3’ UTR（非翻译区域）。
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。
这类载体的转染是稳定转染。
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。
衣藻细胞应该是深绿色。浅绿色或无色（也可能是沿管侧面有一些液滴）细胞表示经过冻融或温度波动，而衣藻细胞对此极为敏感。
衣藻具有无细胞壁和有细胞壁两种类型；137c有细胞壁。
我们提供的是莱茵衣藻（Chlamydomonas reinhardtii）137c株，它被认为是野生型实验室株，交配类型为“mt +”。
衣藻细胞保存6个月后不会丧失活力。据估计，衣藻细胞的保存时间可能达到更久，但我们尚未数据证明保存一年后会丧失活力。
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。
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。
Please see the following suggestions:
1. For best results, the algae need to be between 1 x 10e6 and 2 x 10e6 cells/mL (thus, an OD of 0.3-0.5). The concentration of the cells should not exceed 3 x 106 cells/mL. Cell growth can be measured by OD750 and the linear range will be between 0.2 and 1.2 (in 1 cm lightpass). If the OD is out of the linear range, please dilute the cells and measure again to get an accurate reading.
The formula is: cell number = (OD750 - 0.088)/9 million/mL
2. Transformation of linearized DNA is much more efficient (~70% more efficient) than transformation of circular DNA.
3. The quality and concentration of the DNA are critical to the transformation efficiency. You will want to use PureLink HQ, PureLink HiPure, or equivalent plasmid purification kits for pure DNA. It is better to have a small volume of concentrated pure DNA than a large volume of DNA of low concentration. (If you are transforming linearized plasmid, then after the linearization you will need to clean the plasmid up using either gel extraction or a PCR cleanup kit prior to transformation.) We recommend using 2 µg of linearized plasmid DNA per electroporation.
4. Insertion of the plasmid DNA into the genome occurs randomly. On average, only 20% of transformants will express the gene of interest at appreciable levels. We recommend first screening the colonies by colony PCR to ensure full integration of the promoter and gene of interest, followed by the screening of several positive clones for the expression of the gene of interest to pick the highest expressing clone.
5. Because the C. reinhardtii genome has a very high GC content (~62% GC), the expression levels of recombinant genes are significantly improved if the gene of interest is adapted to the preferred codon usage of highly expressed C. reinhardtii genes.
6. Since the transformation efficiency depends on the random integration of the construct into the genome, the results of the electroporation will depend on the nature of the gene of interest. You can try to transform the control vector that comes with the kit to confirm that the kit and their electroporation method are working correctly.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
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. Our 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.
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 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.
The pChlamy_1 vector does not have the stop codon and the 3' UTR. The pChlamy_3 vector has the 3'UTR that has been shown to increase protein expression.
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.
Yes, the pChlamy_3 vector contains a 3' UTR after the multiple cloning site.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Yes, if you plan to use the pChlamy_1 vector to express high levels of recombinant protein, your insert also needs to contain a 3' UTR (untranslated region) immediately following the stop codon.
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.
Transfection with these vectors is a stable transfection.
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 Chlamydomonas cells should be dark green in color. Light green or even colorless (and maybe some droplets along the side of the vial) cells are indicative of freeze/thawing or fluctuations in temperature, which Chlamydomonas cells are extremely sensitive to.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
There are both cell wall minus and positive strains; 137c has a cell wall.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
We no longer offer any strains of Chlamydomonas. Our reagents and protocols were developed using Chlamydonas reinhardtii 137c. This is considered to be a wild type lab strain, mating type “mt +.”
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
After 6 months of storage, the Chlamydomonas cells did not lose their competency. It is expected that they may with longer storage times, but we have yet to gather data points for loss of competency after one year of storage.
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.
It is a stable transfection.
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.
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.
We recommend the following guidelines:
- For high transformation efficiencies, the OD750 of the C. reinhardtii culture should be > 0.8 before electroporation.
- Carry out all transformation steps at 4 degrees C using solutions pre-equilibrated at 4 degrees C.
- Electroporate the cells using the 100 µL Neon tip in TAP-40 mM sucrose solution or the MAX Efficiency Transformation Reagent For Algae (Cat. no. A24229) at 4 degrees C.
- Use the following Neon electroporation parameters: 2300 volts (Voltage), 13 ms (Pulse Width), 3 (Pulse Number)
For detailed instructions on using the Neon Transfection System, refer to the Neon Transfection System User Guide (https://tools.thermofisher.com/content/sfs/manuals/neon_device_man.pdf).
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.