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View additional product information for GeneArt® Chlamydomonas Protein Expression Kit - FAQs (A24244)
23 product FAQs found
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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.
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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.