Part:BBa_K332011:Experience

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Applications of BBa_K332011

(I) Culture Bti.

Bacillus thuringiensis subsp. Israelensis (BCRC15860)

1. Prepare agar plate for Bti.

  Beef extract  3.0g
  Peptone       5.0g
  Agar          15.0g
  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

  Template DNA（10ng/μl）		    2.0
  B-taq buffer			            5.0
  dNTP（2mM）			            5.0
  forward primer（10μM）		            1.5
  reverse primer（10μM）		            1.5
  B-taq plus DNA polymerase（2Kb）           1.0
  ddH2O		                            34.0  
  Total				            50 μl

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 ACTACTTTAGTAACGGATT

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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