Conjugative DNA transfer between bacteria and the heterokont alga Nannochloropsis
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The heterokont microalgae Nannochloropsis have been the subject of manystudies that focus on genomic and metabolic engineering. Research on thisgenus has been motivated by the algae s high-level production of lipids and inparticular polyunsaturated fatty acids. The work presented in this thesis is partof the effort to develop new tools that enable the rapid and reliable generationof 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 growthof N. oceanica. Transkingdom conjugation has recently been established as a method for transferof DNA from Escherichia coli to two species of heterokont algae (Phaeodactylumtricornutum and Thalassiosira pseudonana). For this phylum of algae, conju-gation has the potential to replace the standard methods of transformation byparticle 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, plasmidtransfer mediated by DH10B/pTA-Mob resulted in the highest conjugationefficiency. Preliminary work identified, that co-incubation of the recipient and donor cells at30 ◦ C was crucial for establishing conjugation. Various conditions and parameterswere investigated for their effect on conjugation efficiency. The parameter ofrecipient to donor ratio cells was found to be an important factor, but withinconclusive results, further research is needed. During co-incubation of recipientand donor cells, dark conditions resulted in higher conjugation efficiencies thanwhen cells were exposed to light. Temporal variations of the co-incubationidentified a maximum for conjugation efficiency at around 90 minutes. A set of novel plasmids, pAPA0602 and pAPA0169, containing endogenouspromoters with reported high expression levels in N. oceanica and the standardplasmid pSELECT100 were used in the conjugal transfer. ALl three of thesebase vectors were gentically altered to include the origin of transfer, oriT. Twoadditional constructs were created from the pAPA-plasmids, which included theyeast sequence, CEN6-ARSH4-HIS3, that has been reported to enable episomal plasmid replication in diatoms. Of these five constructs, the highest conjugationefficiency was observed with pAPA0169. Transformation of N. oceanica by electroporation was conducted to comparetransformation 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 withpAPA0602 and pAPA0169, to evaluate the tolerance against zeocin conferredby a zeocin resistance gene under control of the pAPA plasmid-promoters. Theplasmids, pAPA0602 and pAPA0169 conferred tolerance to the antibiotic atsimilar levels. Overall efficiencies obtained for pAPA0169 and pAPA0602 in thisproject were higher than previously reported numbers. The optimized conjugation protocol, together with the developed plasmids andimproved growth medium for mutant generation, extends the molecular toolboxfor N. oceanica.