Conjugative DNA transfer between bacteria and the heterokont alga Nannochloropsis

Abstract

The heterokont microalgae Nannochloropsis have been the subject of many studies that focus on genomic and metabolic engineering. Research on this genus has been motivated by the algae s high-level production of lipids and in particular polyunsaturated fatty acids. The work presented in this thesis is part of the effort to develop new tools that enable the rapid and reliable generation of Nannochloropsis oceanica mutants.

A new growth medium was developed and evaluated against established Nan- nochloropsis growth media. Compared with previously described growth media, the new medium can be prepared without filtered sea water and improved growth of N. oceanica.

Transkingdom conjugation has recently been established as a method for transfer of DNA from Escherichia coli to two species of heterokont algae (Phaeodactylum tricornutum and Thalassiosira pseudonana). For this phylum of algae, conju- gation has the potential to replace the standard methods of transformation by particle bombardment and electroporation. This project focuses on the develop- ment of transkingdom conjugation as a method of establishing mutants of N. oceanica (CCMP1779). Two E. coli strains were used as donors in the project, S17-1 which has chromosomally integrated transfer functions, and DH10B har- bouring the mobilization plasmid, pTA-Mob. Of these two donor strain, plasmid transfer mediated by DH10B/pTA-Mob resulted in the highest conjugation efficiency.

Preliminary work identified, that co-incubation of the recipient and donor cells at 30 ◦ C was crucial for establishing conjugation. Various conditions and parameters were investigated for their effect on conjugation efficiency. The parameter of recipient to donor ratio cells was found to be an important factor, but with inconclusive results, further research is needed. During co-incubation of recipient and donor cells, dark conditions resulted in higher conjugation efficiencies than when cells were exposed to light. Temporal variations of the co-incubation identified a maximum for conjugation efficiency at around 90 minutes.

A set of novel plasmids, pAPA0602 and pAPA0169, containing endogenous promoters with reported high expression levels in N. oceanica and the standard plasmid pSELECT100 were used in the conjugal transfer. ALl three of these base vectors were gentically altered to include the origin of transfer, oriT. Two additional constructs were created from the pAPA-plasmids, which included the yeast sequence, CEN6-ARSH4-HIS3, that has been reported to enable episomal plasmid replication in diatoms. Of these five constructs, the highest conjugation efficiency was observed with pAPA0169.

Transformation of N. oceanica by electroporation was conducted to compare transformation efficiencies with conjugation efficiencies. The highest transfor- mation efficiency obtained by electroporation was with the plasmid pAPA0602. Electroporation was also used as the method to transform N. oceanica with pAPA0602 and pAPA0169, to evaluate the tolerance against zeocin conferred by a zeocin resistance gene under control of the pAPA plasmid-promoters. The plasmids, pAPA0602 and pAPA0169 conferred tolerance to the antibiotic at similar levels. Overall efficiencies obtained for pAPA0169 and pAPA0602 in this project were higher than previously reported numbers.

The optimized conjugation protocol, together with the developed plasmids and improved growth medium for mutant generation, extends the molecular toolbox for N. oceanica.