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.
