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GeneArt™ Chlamydomonas Protein Expression Vector - FAQs

GeneArt™ Chlamydomonas Protein Expression Vector - FAQs

View additional product information for GeneArt™ Chlamydomonas Protein Expression Vector - FAQs (A24231)

10 product FAQs found

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.

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.

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.

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 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.