TAP Growth Media, optimized for Chlamydomonas culture, 6 x 1 L - FAQs

View additional product information for TAP Growth Media, optimized for Chlamydomonas culture - FAQs (A1379801, A1379802)

22 product FAQs found

I am using the MAX Efficiency Transformation Reagent for Algae to transform Chlamydomonas. However, my efficiency is very low. Any suggestions on how to improve this?

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.

I'm getting low expression due to gene silencing using the pChlamy_1 or pChlamy_3 vector. What do you suggest I try?

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.

What is the difference between pChlamy_4 in the Chlamydomonas Protein Expression kit compared to pChlamy_1 or pChlamy_3 in the engineering kits?

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.

Should I clone and insert not only the coding sequence but also the 3'UTR of my gene of interest?

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.

In the Chlamydomonas kit, there is already an ATG in the vector; does the insert need to be in frame with the ATG in the vector?

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.

Do the pChlamy_3 vectors contain a termination sequence or a 3' UTR for your gene of interest?

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.

I have an older version of the pChlamy vector, pChlamy_1. Do you recommend addition of a 3'UTR to the insert?

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.

What is the expected time constant during transformation?

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.

What is the expected transformation efficiency?

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.

What is the mechanism of integration with the Chlamydomonas engineering kits?

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.

Is transfection with your pChlamy vectors transient or stable?

Transfection with these vectors is a stable transfection.

Can green algae other than Chlamydomonas be transformed with pChlamy vectors?

Unfortunately, we have not yet tested other algae.

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

How can I store my positively selected Chlamydomonas for long-term storage?

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.

What should the cells look like upon arrival?

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.

Does Chlamydomonas have a cell wall?

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.

What is the strain of the Chlamydomonas cells you offer?

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.

What is the shelf life/competency of the Chlamydomonas cells?

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.

What is the formulation of your TAP media?

We offer premade TAP growth media optimized for Chlamydomonas (Cat. No. A1379801). Please see the formulation below:

TAP Media
EDTA (Disodium Salt), EDTA, 0.005%, CAS: 60-00-4
Zinc Sulphate Heptahydrate, ZnSO4-7H2O, 0.002%, CAS: 7446-20-0
Boric Acid, H3BO3, 0.001%, CAS: 10043-35-3
Manganese Chloride Tetrahydrate, MnCl2-4H2O, 0.005%, CAS: 1/5/7773
Cobalt Chloride Hexahydrate, CoCl2-6H2O, 0.002%, CAS: 7791-13-1
Copper Sulphate Pentahydrate, CuSO4·5H2O, 0.002%, CAS: 7758-98-7
Ammonium heptamolybdate, (NH4)6Mo7O24·4H2O, 0.001%, CAS:12027-67-7
Iron(II) Sulphate, FeSO4·7H2O, 0.005%, CAS:7720-78-7
Potassium Phosphate Dibasic, K2HPO4, 0.011%, CAS:11/4/7758
Potassium dihydrogen phosphate, KH2PO4, 0.005%, CAS:7778-77-0
Ammonium chloride, NH4Cl, 0.038%, CAS:12125-02-9
Magnesium Sulfate Heptahydrate, MgSO4 . 7H2O, 0.010%, CAS:10034-99-8
Calcium Chloride Dihydrate, CaCl2 . 2H2O, 0.005%, CAS:10035-04-8
Tris, (HOCH2)3CNH2, 0.242%, CAS:77-86-1
Glacial Acetic Acid, CH3COOH, 0.11%, CAS:64-19-7

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

Is the TAP media good for growing any other kind of green algae?

Unfortunately, we have not tested TAP media with other kinds of green algae. There is an indication that Chlorella will grow if the TAP media is vitamin-fortified, but we don't have the specifics.

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

What type of algae can the MAX Efficiency Transformation Reagent for Algae be used for?

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.

What type of algae can the GeneArt Cryopreservation Kit for Algae be used for?

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.

What does the GeneArt Chlamydomonas Protein Expression Vector include?

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.