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 (<37C), tetR begins to degrade, resulting in the promoter PtetR (BBa_R0011) expressing downstream genes, cry11Aa (BBa_K332012) and Green Fluorescent Protein (GFP)(BBa_E0040).

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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

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Result

Cry11Aa gene of Bacillus thuringiensis subsp. Israelensis was cloned successfully and SpeI and EcoRI sites were found in this gene. To modify it become a standard biobrick, we designed two pair point mutation primers to destroy the restriction enzyme sites (SpeI and EcoRI). Then, this gene was re-amplified using standard biobrick primes and was ligated in backbones (Psb1C3). Cry11Aa gene is one of the useful parts we provided to iGEM.

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).

Link to Cyr11Aa sequence

 

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