Algae Expression Support—Troubleshooting

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.

The promoter for pSyn_1 is the P_sc. P_sc is a weak constitutive promoter that drives basal expression of your gene of interest. The promoter for pSyn_6, on the other hand, is the psbA promoter (P_psbA). P_psbA is a strong constitutive promoter of the psbA gene (encoding photosystem II protein D1) from Synechococcus elongatus, driving the high level expression of your gene of interest.  

Please see the following suggestions:

1.     For best results, the algae need to be between 1 x 10⁶ and 2 x 10⁶ 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.