The first step in transformating any organism is getting the foreign DNA inside the cell. For organisms with a cell wall, methods must be devised to either remove or permeabilize the wall, or to get DNA into the cell through the intact wall. Bacterial cell walls do not seem to represent a significant barrier to DNA uptake, and can be induced to take up foreign DNA simply by being washed in low osmotic medium and glycerol, followed by a brief heat shock. Cell walls can be removed enzymatically from yeast cells to form spheroplasts, or from plant cells to form protoplasts. These wall-less cells can be induced to take up DNA by chemically permeabilizing the cell membrane with polyethylene glycol and/or calcium. Alternatively, DNA can enter yeast spheroplasts or plant protoplasts via electroporation, a method in which a rapid, high voltage electric pulse is used to produce transient pores in a cell membrane.

Based on their research backgrounds, NREL researchers tended to view microalgae as either single cell plants, or pigmented yeasts. In either case, the initial tendency was to try to produce wall-less algal cells as targets for transformation. There had previously been some reports of protoplast production in green microalgae of the genus Chlorella (Braun and Aach 1975; Berliner 1977). NREL researcher Eric Jarvis decided to attempt to introduce foreign DNA into Chlorella protoplasts, with the eventual goal of adapting these protocols for other algal strains with biodiesel production potential.

The production of a stably transformed line of cells involves several steps, including introducing the foreign DNA into the target cell, expressing the foreign gene, stabilizating (replicating) the new DNA by the host cell, and survival and proliferation of the genetically altered cells. Transient expression assays can be used to monitor and optimize just the first two of these processes, i. e., DNA entry and expressing a foreign gene in a population of cells, and thus can be useful intermediate steps in developing genetic transformation systems. Transient assays usually involve the introduction of a gene that codes for an enzyme detectable by a simple biochemical assay (often referred to as a reporter gene). Dr. Jarvis decided to use one such gene, the firefly luciferase gene, to monitor the entry and expression of foreign DNA into Chlorella protoplasts.

The alga used for these studies was C. ellipsoidea (strain CCAP 211/1 a, obtained from the Culture Collection of Algae and Protozoa, Freshwater Biological Association, United Kingdom). Protoplasts were produced using a protocol adapted from Global and Aach (1985). The cells were grown to early stationary phase, then incubated overnight in 10 mg/mL Cellulysin, a crude commercial preparation of the cellulose-degrading enzyme cellulase. Protoplast production was monitored by sonication of the treated cells in water; generally about 80% of the cells were disrupted by this treatment and were considered to be protoplasts. A plasmid containing the luciferase gene driven by plant regulatory sequences was introduced in the protoplasts by mixing the cells with the plasmid DNA for 30 minutes in the presence of 50 mM CaCl2 and 13% polyethylene glycol (mw 4000). The cells were washed and incubated in a regeneration medium overnight. The cells were then harvested and luciferase activity was monitored in crude protein extracts. Luciferase catalyzes the oxidation of luciferin with the production of a photon of light via the following reaction:

luciferase, Mg2+, O2

LUCIFERIN + ATP………………………. > OXYLUCIFERIN + AMP + CO2 + hv

The light produced can be monitored using a scintillation counter or a luminometer.

The results of these experiments are shown in Figure II. B.6. Luciferase activity was detectable in protoplasts treated with the luciferase plasmid, but not in protoplasts that had not been exposed to plasmid or to polyethylene glycol. Intact cells did not take up the DNA. There was a significant decrease in luciferase expression when carrier DNA was left out of the transformation reaction (“carrier DNA” is usually sheared genomic DNA from calf thymus or salmon sperm that is added to reduce the effects of cellular nucleases on the added plasmid DNA). Monitoring of the luciferase activity over time showed that the activity was maximal at about 24 hours after exposing the protoplasts to the plasmid; expression decreased over time and was virtually undetectable after 80-100 hours. Unfortunately, attempts to regenerate the protoplasts into viable walled cells were unsuccessful.

These results were important as they demonstrated the first successful steps in developing a genetic transformation system for microalga, including the production of viable protoplasts, the introduction of DNA into the protoplasts, and the expression of a foreign gene by the algal cells. This last point was very significant, as homologous genes were required to achieve transformation in another green alga (Chlamydomonas). The dogma in the field was that heterologous gene expression in green algae would likely be unsuccessful due to codon biases resulting from high GC contents. The work resulted in a publication (Jarvis and Brown 1991), and was the basis for later studies in which the luciferase gene was used to monitor promoter activities in Cyclotella (discussed later). However, attempts to adapt this procedure to algal strains with significance to the biodiesel project were unsuccessful. The composition of microalgal cell walls is highly variable between species and even between isolates of the same species. Some unsuccessful efforts were made to determine the enzymatic conditions for wall degradation for several oleaginous algal strains. However, the conclusion, in the words of the

project manager at the time, was that this was “an endless pit of fruitless endeavor”, and the decision was made to explore other methods of introducing DNA into microalgal cells. In addition, although low levels of luciferase expression were acheived in Chlorella, the decision was made to pursue the development of selectable marker systems that would allow the isolation of very rare individual transformants within a population of microalgal cells. This will be discussed in the following section.