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3.6 Transformation of pYES2-Cry1Ac22-EGFP
into
S. Cerevisiae
The
Saccharomyces cerevisiae
competent cells were
obtained through the method of LiAc/SS2
DNA/PEG according to Invitrogen. Add 500 μL
competent cells into 100 μL Buffer 1. mixed with
1~2 μg recombinant plasmids identified positive
and 2 μL characinlike DNA, shake lightly. Then add
600 μL Buffer 2 and shaked for 30 min at 30
℃
.
After that, add 70 μL DMSO and treated at 42
℃
for 15min. Centrifugated the solution for 10 s, add
300~500 μL TE Buffer to wash and precipitate for
30 min at 30
℃
, spread on SD-ura plate, and
cultured for 3 days at 30
℃
. The bacterial colony
become transformant recon; The bacterial colony
was picked and observed under microscope for
further identification.
3.7 The induced expression of recombinant
plasmid in
Saccharomyces Cerivisiae
The positive colony was vaccinated into SC-U fluid
medium with 1% raffinose and cultured at 170 r/min
and 30
℃
for overnight. The product was centri-
fuged by 12 000 r/min at 4
℃
for 5 min. The thallus
were precipitated by SC-U fluid medium with 2%
galactose until its OD
600
becomes 0.4, then cultured
under the condition of 30
℃
,170 r/min. Collect
galactose to culture bacterial liquid for 6 h, 12 h,
24 h, then grind it by liquid nitrogen. After that,
collect supernatant fluid for SDS-PAGE, and
identify it by 10% PAGE.
3.8 The localization of
cry1Ac22
gene in yeast cell
The bacterial colony was transferred from plate into
SC screening medium, cultured at 30
℃
for
overnight. 50% of the solution was washed 3 times,
then transferred to SC induction medium (20%
galactose instead of glucose) and cultured for 20 h.
Collect induced and non-induced thallus, wash 3
times, add 2% low-gelling temperature agarose,
shake slightly, and placed on microscope slide
pretreated at 50
℃
, and then placed in dark for 5 min,
followed by observation under fluorescence
microscope.
Authors’ contributions
Shenkui Liu and Zhuoming Liu design and execute this experiment.
Youzhi Li participates the experiment design and data analysis; Xuanjun
Fang is the person in charge of this project, conducting experiment
design, data analysis, writing and modifying of the manuscript. All
authors have read and approved the final manuscript.
Acknowledgements
This research is funded by the project of China National Bt Collection
Initiative and National 863 plans (Project No. 2004AA2111112). Authors
appreciate Dr Xinxin Zhang from the ASNESC of Northeast Forestry
University, Mr Wenfei Zhang and Liu Xie from HITAR for technological
supports and helpful advice on the experiment. Thanks for peer
reviewers for there useful advice and revising suggestion to this paper.
And we mentioned some reagent suppliers and sequencing suppliers in
this work, that doesn’t mean we would like to recommend or endorse
their products and services.
References
Adang M.J., Staver M.J., Rocheleau T.A., Leighton J., Barker R.F., and
Thompson D.V., 1985, Characterized full length and truncated
plasmid clones of the crystal protein of Bacillus thuringiensis subsp.
kurstaki HD-73 and their toxicity to Manduca sexta, Gene, 36(3):
289-300 doi:10.1016/0378-1119(85)90184-2
Knowles B.H., and Ellar D.J., 1986, Characterization and partial
purification of a plasma membrane receptor for Bacillus
thuringiensis δ-endotoxin with different insect specificity,
Biochimica et Biophysica Acta, 924: 509-518
Li J., Carrol J., and Ellar D.J., 1991, Crystal structure of insecticidal
δ-endotoxin from Bacillus thuringiensis at 2.5Å resolution, Nature,
353(6347): 815-821 doi:10.1038/353815a0 PMid:1658659
Liu Z.M., Liu S.K., Li Y.Z., and Fang X.J., 2010, Expression and
purification of
Bacillus thuringiensis
Cry1Ac22 protein in
Escherichia coli, Bioscience Methods, Vol. 1, No. 2 (doi:
10.5376/bm.2010.01.0002)
Misteli T., and Spector D.L., 1997, Applications of the green fluorescent
protein in cell biology and biotechnology. Nat. Biotechnol., 15(10):
961-964 doi:10.1038/nbt1097-961 PMid:9335045
Sambrook J.E., Fritsch F., and Maaiatis T., 2002, Experiment guidance of
Molecular clone, Science press, Beijing, China, pp.483-485
Xie L., Zhang W.F., Liu Z.M., Cai Y.G., Li Y.Z., and Fang X.J., 2010,
Characterization of a new highly toxic isolate of Bacillus
thuringiensis from the diapausing larvae of silkworm and
identification of cry1A 22 gene, Bt Research, Vol.1 No.1 (doi:
10.5376/bt.2010.01.0001)