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Bt Research (Online) 2010, Vol.1 No.2
http://bt.sophiapublisher.com
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Table 1 Strans and plasmids used in this study
Strains and plasmids Characteristics Origin
B. thuringiensis
Bt W015- 1 Wild strain of B. thuringiensis This lab
Bt subsp. kurstaki HD73 Model strain of B. thuringiensis haboring cry1Ac1 gene This lab
Bt subsp. kurstaki HD1 Model strain of B. thuringiensis This lab
Bt subsp. israelensis AND508 Mutant is a derivative of AND406 cured of pTX14-2 and
pTX14-3
This lab
Escherichia coli
E. coli JM110 Dam, dcm, supE44, hsdR17, thi, leu, rpsL1, lacY galK, galT,
aratonA thr, tsx, D (lac-proAB)
(F0, traD36, proAB, lacI q ZDM15)
Novagen
E. coli M15 Nals, Str s, Rifs, Thi-, Lac -, Ara +, Gal+, Mtl-, F-, RecA+ , Uvr+ , Lon+
with KanaR
QIAGEN
Plasmid
pMD18- T AmpR, AT-easy clone vector Takara
pMDK2 AmpR pMD18 -T vector harboring PCR fragment by primer pairs
of K5un2/K3un2
This study
pMDK3 AmpR pMD18 -T vector harboring PCR fragment by primer pairs
of K5un3/K3un3
This study
pMDIS AmpR pMD18 -T vector harboring PCR fragment by primer pairs
of cry1I5/cry1I3
This study
pQE -30 AmpR, T5 promotor, expression vector QIAGEN
pQE1Ac22 AmpR, pQE- 30 vector carrying cry1Ac22 gene This study
Table 2 Primers for gene identifiing, cloning and expression
Primer name Sequence Origin and reference
K5un2 AGGACCAGGATTTACAGGAGG
K3un2 GCTGTGACACGAAGGATATAGCCAC
Kuo and Chak (1996)
K5un3 CAATGCGTACCTTACAATTGTTTAAGTAAT
K3un3 CCTCCTGTAAATCCTGGTCCT
Kuo and Chak (1996)
cry 1I5 TTGCGTTAGCGACAAGGAAAT
cry 1I3 AATGTGCCAGGTACGGGTTC
This study
E1A5 CGGGATCC AGAGATGGAGGTAACTTATG
E1A3 ACGCGTCGAC TGAGACTATTCCTCCATAAG
This study
2.4 Observation of parasporal crystal by Scanning
Electron Microscope (SEM)
Bt strains were grown in BP medium at 30℃ for 3
days until sporulation was complete as examined by
light microscope with an oil-immersion lens. The
spores and crystals were collected by centrifugation
at 4℃ at 12,000 g for 10 min, and the precipitate was
washed three times with ice-cold sterilized
double-distilled water. The spore-crystal
suspensions were placed on aluminum mount and
fixed in 1% OsO4 after the samples were air-dried
overnight. The samples were then coated with gold
in an IB- 5 ion coater (HITACHI, Japan). The SEM
observation was conducted on a HITACHI
S-3400N (HITACHI Japan) at a voltage 15 kv
following the machine instructions for the devise
(Zhang et al., 2009).
2.5 Plasmids profiles of Bt strains
Bt W015-1 was grown to the final OD600 value of
2.0 at 30℃ in LB medium with shaking at 220 r/min.
The strain cells were pelleted by centrifugation at
10, 000 g for 5 min and re-suspended in GTE buffer
[2.5 mmol/L Glucose, 25 mmol/L Tris-Cl, 10 mmol/L