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Bt Research (Online) 2010, Vol.1 No.3
http://bt.sophiapublisher.com
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3 Materials and Method
3.1 Plasmid and Strains
Strains and plasmids used in this study were stored
in Haide Institute of Tropical Agricultural
Resources (HITAR) (Table 1).
Table 1 Strains and plasmids used in this research
Strains and plasmids Characterization Origin References
pMD18-T-Cry1Ac22 AmpR, pMD18-T carrying cry1Ac22 gene This research Xie et al., 2010
pGFP AmpR, pUC ori, Plac, MCS HITAR Unpublished
pYES2 AmpR ,GAL1 promoter, T7 promoter, MCS, CYC1
transcription terminator, URA3 gene F1 origin
HITAR Unpublished
E. coli JM109 E. coli recA1 supE44 endA1 hsdR17 gyr A96 relA1
thi△(lac-proAB)
HITAR Sambrook et al., 2002
INVScl yeast strains Genotype: MATa his3△1 leu2 trp1- 289 ura3- 52
Phenotype: His-, Leu-, Trp-, Ura-
HITAR Unpublished
3.2 Culture media
The culture media for yeast growth is SD: yeast
nitrogen-containing bases 0.67%, glucose 2.00%,
compounds derived from an-uracil amine acid
0.13%; The culture media for inducing the
expression of GFP is SC (yeast nitrogen-containing
bases 0.67%, Galactose 2.00%, compounds derives
from an-uracil amine acid 0.13%). LB culture
media was used for E. coli, according to Sambrook
et al (2002).
3.3 Reagents and devices
Yeast nitrogen base without amino acids is from
Difco. Restriction enzymes, Taq DNA polymerase,
denatured characinlike Single-Stranded DNA, dNTP
were bought from TaKaRa. Lysozyme, RNaseA,
Recycling kit of DNA agarose gel, Amino Acids,
DMSO, PEG3350, Raffinose, Galactose were
bought from Amresco. Tris (pH 8.0) phenol,
chloroform and such kind of reagents were national
A.R. PCR instrument used in this study is ABI 9600.
Perpendicular-plate-electrophoretic apparatus is
products of USA Bio-Rad firm. Microscope
Olympus BX50 is used for fluorescence observation.
3.4 The construction of recombinant plasmid
pGFP-Cry1Ac22
The pGFP vector and pMD18-T-Cry1Ac22 vector
were digested by BamHⅠ and KpnⅠ . The products
were separated by 1.0% agarose gel electrophoresis.
The target fragments were recycled by DNA
agarose gel recycling kit, and were ligated with
vector at 16℃ for overnight. 4 μL of the ligation
products were added into E. coli JM109 competent
cells (kept on ice). The tube was flicked slightly for
uniform distribution, kept on ice for 30 min, then
kept at 42℃for 90 s for thermal shock, and then add
into 400 μL LB culture media, followed by Shaking
culture at 37℃for 1h. Then 100 μL broth was spread
on LB screening plate (containing 100 μg/mL
ampicillin), and culture at 37℃ for overnight. The
colony plasmid was extracted using boiling method
and the identification of positive clones was
performed through enzyme digestion.
3.5 The construction of recombinant plasmid
pYES2 -Cry1Ac22-EGFP
The pGFP-Cry1Ac22 identified positive and pYES2
were digested by BamHⅠ and NotⅠ for 3 h at
37℃. Separated the products by 1.0% agarose gel
electrophoresis, recycled the target fragments using
DNA agarose gel recycling kit, and ligated the two
target fragments with vector at 4 ℃ for overnight.
Insert the ligation products into E. coli JM109
competent cells, and choose the positive bacterial
colony for expanding and extracting recombinant
plasmid. The identification of positive clones was
performed by enzyme digestion.