Toward a genetic transformation system for the marine microalga Emiliania huxleyi
the marine microalga Emiliania huxleyi
Jan Strauss*, Katja Metfies, Klaus Valentin
Alfred-Wegener-Institute for Polar and Marine Research, Bremerhaven, Germany
transgenic E. huxleyi genefunction ?
gene
physiological characterization +
expression vector
wild-type
A start
The aim of this work was to establish a transformation system for the cosmopolitan coccolithophoridEmiliania huxleyi by accomplishing the following mandatory methods:
Cultivation on agar plates.
R lt
transformation
genetic
transformation gene function
genome
Integration
Development of a selection method for transformants.
Construction of anE.huxleyi-specific expression vector.
Cloning of a strong promoter to drive gene expression.
Development of a transformation protocol.
Establishing a transformation system will provide a
powerful tool for functional genomics.
Result
E.huxleyican grow on agar plates.
Cells are sensitive to the antibiotics chloramphenicol, cycloheximide, G418 and puromycin
select transformants modified genome
Integration
site
Problems
To keepE.huxleyicultures axenic.
To choose the right selection marker gene and reporter gene.
Develop a feasible methodological approach to select for transformants.
puromycin.
The modified diatom vector using zeocin resistance as selection marker gene was inoperative.
resistent colonies cultivation of single clones Fig.1: E.hux grows on
agar plate.
Conclusion
Providing a methodological approach to select transformants is a key towards the establishment of an E.huxleyi-specific expression vector and transformation system:
Chloramphenicol, cycloheximide, G418, and puromycin and its
T b 1 H tibi ti ff t f E h l i
analysis
p y p y
respective resistance genes are of high potential use.
G418 and its respective resistance gene is proposed to be most promising as it is already used in microalgae transformation systems[1,2].
Feasibility of the biolistic approach[3]to transformE.huxleyi remains to be shown.
antibiotic growth concentration
Kanamycin + 1 mg/mL
Streptomycin + 1 mg/mL
Zeocin + 1 mg/mL
Hygromycin B + 1 mg/mL
Tab. 1: How antibiotics effect of E.huxleyi.
growth rate [d-1]
0,2 0,4 0,6 0,8 1,0 1,2
DNA RNA Protein
Future Perspective
Map regulatory sequences (promoters).
Design functional expression vector cassette.
Develop a new transformation vector.
Do further transformation experiments with a gene gun.
Phleomycin + 500 µg/mL
Blasticidin + 200 µg/mL
G418 - 500 µg/mL
Choramphenicol - 100 µg/mL
Puromycin - 50 µg/mL
C l h i id 1 / L
Fig. 3: Methodological approach
control
mycin, 1000 µg/mL mycin, 1000 µg/mL
ticidin, 200 µg/mL eocin, 1000 µg/mL
om ycin, 500 µg/mL
mycin, 1000 µg/mL omy
cin, 50 µg/mL henicol, 100 µg/mL
G418, 500 µg/mL heximide, 1 µg/mL -0,4
-0,2 0,0 0,2
Acknowledgements
Thanks to the Sea Ice Group at AWI and A. Gruber (University of Konstanz) for providing a diatom vector.
This work was supported by:
p g g
References
[1]Dunahay et al. (1995) J Phycol31:1004-12; [2]Zaslavskaia et al. (2000) J Phycol36:379-86;
[3]Sanford et al. (1993) Method Enzymol217:483-509 Cycloheximide - 1 µg/mL
*Current address
School of Environmental Sciences University of East Anglia, Norwich, UK Email: J.Strauss@uea.ac.uk
Fig. 2: Growth or motality rates of E.huxleyi treated with antibiotics.
Kanam y
Streptomy Blasti
Zeo Phleom
Hygromy Puro Chloram
phe Cyclohe
