"
Team:HKUST/Group4
From 2009.igem.org
(Difference between revisions)
| Line 59: | Line 59: | ||
<img src="http://igem2009hkust.fileave.com/wiki/Group4/figure1.jpg " width=450; height=90 /></a><br> | <img src="http://igem2009hkust.fileave.com/wiki/Group4/figure1.jpg " width=450; height=90 /></a><br> | ||
| - | (2) Only BinA gene is fused with GST, cloned into the same multi-cloning site of pESC-Leu expression vector under the | + | (2) Only BinA gene is fused with GST, cloned into the same multi-cloning site of pESC-Leu expression vector under the GAL10 promoter.</p> |
<img src="http://igem2009hkust.fileave.com/wiki/Group4/figure2.jpg " width=450; height=90 /></a><br> | <img src="http://igem2009hkust.fileave.com/wiki/Group4/figure2.jpg " width=450; height=90 /></a><br> | ||
| - | (3) Only BinB gene is fused with GST, cloned into the same multi-cloning site of pESC-Leu expression vector under the | + | (3) Only BinB gene is fused with GST, cloned into the same multi-cloning site of pESC-Leu expression vector under the GAL1 promoter.</p> |
<img src="http://igem2009hkust.fileave.com/wiki/Group4/figure3.jpg " width=450; height=90 /></a><br> | <img src="http://igem2009hkust.fileave.com/wiki/Group4/figure3.jpg " width=450; height=90 /></a><br> | ||
These constructs will be transformed into the yeast and induced by galactose. Then we will use western blot to test the expression of toxin by anti-GST antibody. We can estimate the protein level as well as the yeast phenotype: whether they are resistant to the toxin.</p> | These constructs will be transformed into the yeast and induced by galactose. Then we will use western blot to test the expression of toxin by anti-GST antibody. We can estimate the protein level as well as the yeast phenotype: whether they are resistant to the toxin.</p> | ||
Revision as of 10:04, 20 October 2009
a
Protein Expression
We have designed several constructs to test the binary protein's toxicity to insects as well as to yeast:(1) BinA and BinB genes each fused with GST, cloned into the multi-cloning sites of pESC-Leu expression vector under the GAL10 and GAL1 promoter, respectively.
(2) Only BinA gene is fused with GST, cloned into the same multi-cloning site of pESC-Leu expression vector under the GAL10 promoter.
(3) Only BinB gene is fused with GST, cloned into the same multi-cloning site of pESC-Leu expression vector under the GAL1 promoter.
These constructs will be transformed into the yeast and induced by galactose. Then we will use western blot to test the expression of toxin by anti-GST antibody. We can estimate the protein level as well as the yeast phenotype: whether they are resistant to the toxin.
Drosophila Larvicidal Essay
Next we will test the biological function of toxin produced in yeast to kill Drosophila larvae. The Drosophila larvicidal activity is tested by diluting yeast cells expressing the toxin in water and adding them to water containing 10 larvae in each well of a 24-well tissue culture plate (diameter of the well is 1.5 cm)(Figure 1). Mortality is recorded after incubation at 30°C for 48 h. LC50 is determined by Probit analysis[6].
Fig 1. Drosophila Larvicidal assay. Diluted yeast cells expressing the toxin and 10 larvae are incubated in a 24-well tissue culture plate. Mortality is recorded after incubation is measured. LC50 is determined by Probit analysis.
iGEM 2009
