Despite others’ success with genomic PCR of Dunaliella-specific nucleotide sequences, our efforts to obtain the actin and rbcS2 promoters and nitA 3′-UTR were unsuccessful. Although the products from these PCR attempts appeared to be the correct fragment length, upon sequencing, it became clear that these were not the targets that we set out to amplify. BLAST analysis of some sequences recovered showed relevant homology to genes from Dunaliella viridis and Arabidopsis lyrata, but not the targeted promoters specific to D. salina.

3.3 Discussion

The inefficacy D. salina promoter and 3′-UTR amplification greatly inhibited our efforts to develop and test genetic transformation techniques with this alga. The failed attempts to amplify the actin, rbcS2, and nitA regulatory elements raises some concern for the accuracy of the sequences deposited in GenBank. Another possible cause for this lack of success might come from the strain of D. salina used as a source for these sequences. All of the prior work with these promoters and 3′-UTR has been done using the UTEX 1644 D. salina strain. It is conceivable that the UTEX 1664 sample supplied to us was either misidentified or contaminated. Additionally, the published sequence information might not have actually come from UTEX 1644, as claimed. From our observations, the CCAP 19/18 strain was consistently able to produce P-carotene when cells accumulated and dried on the inner surface of the culture flasks, unlike UTEX 1644. Although, the only way to know the identity of each strain for certain would be to perform genomic analysis of the 18S rRNA. This technique has been established for many species of Dunaliella, including both the UTEX 1664 and CCAP 19/18 strains (Olmos et al., 2000; Polle et al., 2008).

Due to our inability to construct Dunaliella-specific expression vectors, the attempts to genetically transform D. salina were limited to the use of the C. reinhardtii bleomycin- resistance plasmid, pSP124. There is evidence that genetic regulatory sequences from D. salina demonstrate activity in C. reinhardtii (Walker et al., 2004); thus, it is possible that the same is true of C. reinhardtii promoters for use in D. salina. Unfortunately, after numerous trials of electroporation and microparticle bombardment, no viable transformants were recovered after selection on bleocin plates.

It was surmised that the force of impact imposed by gold microparticles would be too much for D. salina, which lacks a cell wall; however, electroporation should have been more accommodating. Testing both high and low voltage (4 and 1 kV cm-1) electroporation conditions as well as high and low cell densities (4 x 107 or 1 x 106 cells ml-1) for transformation proved unacceptable for even transient expression of the ble gene. We did find that, with both methods of transformation, control samples remained viable after the procedure, so at least the electrical pulse itself was not killing the cells. Without endogenous D. salina promoters, we are unable to determine whether the absence of transgene expression was a result of improper transformation conditions or inactive promoters. Notwithstanding the pitfalls encountered during the molecular work with D. salina, experiments pertaining to antibiotic and herbicidal tolerance yielded results that will complement the microbiological understanding of D. salina and aid with future genetic manipulation of the organism.

3.4 Conclusions

Our approach to genetic transformation of a Dunaliella salina will hopefully set the stage for future efforts toward genetic engineering of this organism and, perhaps, act as a template for genetic bioprospecting with other novel algal species. Based on our dosage response experiments, we were able to narrow down the already short list of selective agents applicable to D. salina to the antibiotic bleomycin and the herbicide phosphinothricin and quantify the minimum inhibitory concentrations in both solid and liquid medium.

Limited by insufficient sequence information, we were unable to construct the proposed D. salina transformation vectors and transformation with existing Chlamydomonas vectors proved to be unsuccessful. Dunaliella salina is known to be a delicate organism due to its lack of a cell wall; thus, established transformation techniques may be too forceful for this organism. It is also reasonable to believe that gene silencing is an issue in D. salina and, in addition to optimizing transformation protocols suitable for this alga, molecular methods for promoting stable transgene integration and expression are of great interest to continued work in with Dunaliella species.

Although some accomplishments have been made in the area of D. salina molecular biology, genetic work with this alga warrants additional investigation. In addition to the chloroplast and mitochondrial genomes of D. salina CCAP 19/18, it is anticipated that the recent release of the nuclear genome will greatly encourage further genetic and metabolic engineering of this organism.

In the same way that computers are the coupling of software and hardware, microalgal cultivation systems rely on both the algal organism being grown and the vessel used to amass the cells. While one piece of software can potentially be run on various hardware devices, the two are often developed together and designed accordingly; the same is true with algal culture systems. Whether the growth environment is a raceway pond or a photobioreactor, there exist innumerable prospective algal species that could be cultivated. As the field of microalgal biotechnology moves more toward engineered algae and high- performance PBRs, the unique qualities of the organism will be paired with bioreactor design considerations. Just as the computing power of microchips is always increasing and new versions of operating systems are ever more frequently available, it is expected that algal species that are selected or engineered for high productivity will constantly demand more of their cultivation systems and vice versa.