Contribution to C. reinhardtii Glass Bead Transformation Protocol

Our team used a glass bead transformation protocol detailed by Pei-Hsun Kao and I-Son Ng to transform our plasmid into C. reinhardtii ^[5]. The glass bead transformation method was chosen because of its previously cited success in nuclear transformation, and also because our team did not have access to the proper resources to be able to follow electroporation protocols for C. reinhardtii cells. However, we had little success in our early transformations using the glass bead protocol. Our troubleshooting efforts are detailed below:

Firstly, our initial attempts at transformations were not uniform in the number of cells that were being used. Although many of the glass bead protocols in the literature do not specify a cell density value, our trial and error found that a cell density of 10⁶ cells/mL is ideal for transformation, such that after resuspension, the resulting cell density is 10⁸ cells/mL. Our 60 mL cultures of C. reinhardtii strain CC-400 generally reached 10⁶ cells/mL 5 to 6 days post-inoculation. These numbers are validated by the protocols in Kindle et al. (1990) and the Erb Lab at MPI-Marburg ^[6].

Another unintentional factor that may reduce transformation efficiency is using circular DNA in the transformation rather than linearized DNA. Both approaches have the potential to result in transformants, but our team saw much higher success rates when using a linearized plasmid. To linearize the DNA, assess the plasmid using a visualization platform like Benchling or SnapGene to find an enzyme that has a singular cut site in the entire plasmid. Use this enzyme to perform a restriction digest. Use 1 ng of DNA, and the total reaction volume should balance to 25 uL.

Another crucial step in the transformation process is allowing the algal cells to recover for 24 hours post-transformation. This should be done after vortexing by transferring the cells, PEG, and DNA mixture to a flask containing 30 mL of TAP medium. The flask should be located in a dark area of the lab, or at least one that receives no direct sunlight. After shaking for 24 hours, centrifuge the 30 mL volume down and resuspend in 1/100 volume of TAP media.

To further optimize transformation efficiency, polyethylene glycol (PEG) was added to the glass bead tube before vortexing, and linearized DNA was used in the transformation.

Figure 1. (a) Transformants visible after following Kao and Ng Protocol (b) Transformants visible after following Kao and Ng Protocol with the addition of 20% polyethylene glycol (PEG) to the cells prior to vortexing.

Our glass bead transformation protocol is also available on our Experiments page and in the Phototroph Community Handbook. A video guide made by our team for this protocol is available below:

References