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Team:NCTU Formosa/Cry production
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| + | <p>Bacillus thuringiensis, which is a Gram-positive bacteria. It can produce insecticidal crystal proteins(ICP) which are proteolytically processed by gut proteases into the activated δ-endotoxins. The toxins activated by gut proteases bind to specific binding sites on the brush border membranes of insect midgut epithelial cells. The conformational change in the toxin molecules triggers the insertion of their pore-forming domain into the membrane. Finally, colloid-osmotic swelling and lysis of the cell result in the death of the larvae. </p> | ||
| + | <p>Cry11Aa protein is one of the crystal protein coded in Bacillus thuringiensis subsp. Israelensis and it is highly toxic to certain dipteran larvae, such as Aedes, and Anopheles larvae. The length of Cry11Aa sequence is about 1.9Kb. </p> | ||
| + | <p>In our project, the cry weapon system produce crystal protein, targetting the wrigglers, larvae of mosquitoes. It is controlled by the tetR-repressible promoter PtetR(<a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0040">BBa_R0040</a>), which in turn is regulated by a temperature control system(<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K332031">BBa_K332031</a>). When E.coli is released into environment (<37°C), tetR begins to degrade, resulting in the promoter PtetR(<a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0011">BBa_R0011</a>) expressing downstream genes, cry11Aa(<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K332011">BBa_K332011</a>) and Green Fluorescent Protein(GFP)(<a href="http://partsregistry.org/wiki/index.php/Part:BBa_E0040">BBa_E0040</a>). </p> | ||
| + | <p><span style="font-weight:normal;font-size:200%;line-height:150%"> <a href="http://partsregistry.org/wiki/index.php/Part:BBa_E0040">Link to Cyr11As sequence</a></span></p> | ||
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Revision as of 01:06, 28 October 2010
Wet Lab>Cry production
Outline
The cry weapon system produce crystal protein, targetting the wrigglers, larvae of mosquitoes. The cry11Aa gene is cloned from Bacillus thuringiensis subsp. Israelensis. The cry protein is controlled by the tetR-repressible promoter PtetR (BBa¬_R0040), which in turn is regulated by a temperature control system. When E.coli is released into environment (<37C), tetR begins to degrade, resulting in the promoter PtetR (BBa_R0011) expressing downstream genes, cry11Aa (BBa_K332012) and Green Fluorescent Protein (GFP)(BBa_E0040).
Procedures
(I) Culture Bti. Bacillus thuringiensis subsp. Israelensis (BCRC15860)
1. Prepare agar plate for Bti.Beef extract 3.0g
Peptone 5.0g
Agar 5.0g
ddH2O 1 L
*adjust PH to 7.0
*No antibiotic
*Be aware of contamination
2. Plate Bti. on the medium and incubate the cultures at 30°C overnight.
(II) Clone cry11Aa from Bti. into TA vector
1. Extract genomic DNA of Bti. by liquid nitrogen.
2. Add some ddH2O to dilute the DNA.
3. Design primers
Vf2: ATTCAATAAAAGGTGGAATGAATTATGGA Tm: 53°C
VR: GTGCTAACATGACTTCTACTTTAGT Tm: 52.8°C
4. Find the best PCR condition by gradient PCR. Anneling temperature: 51°C±10°C
5. PCR by B-taq plus on the best condition
6. Digestion to confirm the cry11Aa fragment and enzyme sites.
7. TA clone
8. DNA sequencing
(III) PCR mutagenesis at two enzyme sites --- EcoR1 and Spe1
1. Design primers by primerX.
EcoR1-Vf2: CGG GTA CAA TCT CAG AAC TCG GGA AAT AAT AGA A
EcoR1-VR: TTC TAT TAT TTC CCG AGT TCT GAG ATT GTA CCC G
Spe1-Vf2: ATA ATG AAT GGG GAG GAC TGG TTT ATA AGT TAT TAA TGG G
Spe1-VR: CTT CCC CCA TTA ATA ACT TAT AAA CTA GTC CTC CCC ATT CAT
2. Digestion to confirm the fragments
(IV) PCR construction of Biobrick parts
1. Design primers by Assembly standard 10.
Vf2: GTTTCTTC GAATTC GCGGCCGC T TCTAG ATGGAAGATAGTTCTTTAGATACT
VR: GTTTCTTC CTGCAG CGGCCGC T ACTAGT A CTACTTTAGTAACGGATT
2. PCR condition
3. Ligation to backbones(Psb1C3).
(V) Transform into E.coli
1. Thaw competent cells and BBa_K332012 plasmid on ice.
2. Add 2ul plasmid to competent cell and place in ice for 5 minutes.
3. Put the transformed cells into 42℃ water bath for 45 seconds.
4. Plate the cells on the appropriate media and antibiotic, such as agar plates with 25 µg/ml kanamycin.
5. Incubate the cultures at 37°C overnight.
(VI) Culture with mosquito larvae and observe
Result
Bacillus thuringiensis, which is a Gram-positive bacteria. It can produce insecticidal crystal proteins(ICP) which are proteolytically processed by gut proteases into the activated δ-endotoxins. The toxins activated by gut proteases bind to specific binding sites on the brush border membranes of insect midgut epithelial cells. The conformational change in the toxin molecules triggers the insertion of their pore-forming domain into the membrane. Finally, colloid-osmotic swelling and lysis of the cell result in the death of the larvae.
Cry11Aa protein is one of the crystal protein coded in Bacillus thuringiensis subsp. Israelensis and it is highly toxic to certain dipteran larvae, such as Aedes, and Anopheles larvae. The length of Cry11Aa sequence is about 1.9Kb.
In our project, the cry weapon system produce crystal protein, targetting the wrigglers, larvae of mosquitoes. It is controlled by the tetR-repressible promoter PtetR(BBa_R0040), which in turn is regulated by a temperature control system(BBa_K332031). When E.coli is released into environment (<37°C), tetR begins to degrade, resulting in the promoter PtetR(BBa_R0011) expressing downstream genes, cry11Aa(BBa_K332011) and Green Fluorescent Protein(GFP)(BBa_E0040).
