• norsk
    • English
  • norsk 
    • norsk
    • English
  • Logg inn
Vis innførsel 
  •   Hjem
  • Fakultet for naturvitenskap (NV)
  • Institutt for bioteknologi og matvitenskap
  • Vis innførsel
  •   Hjem
  • Fakultet for naturvitenskap (NV)
  • Institutt for bioteknologi og matvitenskap
  • Vis innførsel
JavaScript is disabled for your browser. Some features of this site may not work without it.

Conjugative DNA transfer between bacteria and the heterokont alga Nannochloropsis

Khider, May Laura Kilano
Master thesis
Thumbnail
Åpne
18271_FULLTEXT.pdf (38.10Mb)
18271_COVER.pdf (1.556Mb)
Permanent lenke
http://hdl.handle.net/11250/2563969
Utgivelsesdato
2018
Metadata
Vis full innførsel
Samlinger
  • Institutt for bioteknologi og matvitenskap [888]
Sammendrag
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.
Utgiver
NTNU

Kontakt oss | Gi tilbakemelding

Personvernerklæring
DSpace software copyright © 2002-2019  DuraSpace

Levert av  Unit
 

 

Bla i

Hele arkivetDelarkiv og samlingerUtgivelsesdatoForfattereTitlerEmneordDokumenttyperTidsskrifterDenne samlingenUtgivelsesdatoForfattereTitlerEmneordDokumenttyperTidsskrifter

Min side

Logg inn

Statistikk

Besøksstatistikk

Kontakt oss | Gi tilbakemelding

Personvernerklæring
DSpace software copyright © 2002-2019  DuraSpace

Levert av  Unit