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

  cry forward:  ATTCAATAAAAGGTGGAATGAATTATGGA       Tm: 53°C
  cry reverse:  GTGCTAACATGACTTCTACTTTAGT           Tm: 52.8°C

4. Find the best PCR condition by gradient PCR.

  Anneling temperature: 51°C±10°C
  
  lane 1: 1Kb marker
  lane 2~13: cry11Aa PCR product(51°C±10°C) length:2Kb

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
   
  94°C   5  min
  94°C  30  sec
  53°C  30  sec
  72°C  2.5 min
  72°C  10  min
  cycles:25

  
  In the last two lanes are cry11Aa gene. The length is about 2Kb.

6. TA cloning

7. Digestion to confirm the cry11Aa fragment and enzyme sites.

  
  lane 1: 1Kb marker
  lane 2: plasmid uncut
  lane 3: Not1 digestion(~2981bp,1966bp)
          [There are two Not1 sites on TA vector.]
  lane 4: EcoR1 digestion(~2997bp,1695bp,237bp)
          [There are two EcoR1 sites on TA vector and another EcoR1 site on cry11Aa.)

8. DNA sequencing

(III) PCR mutagenesis at two enzyme sites --- EcoR1 and Spe1

1. Design primers by primerX.

  EcoR1-f: CGG GTA CAA TCT CAG AAC TCG GGA AAT AAT AGA A
  EcoR1-R: TTC TAT TAT TTC CCG AGT TCT GAG ATT GTA CCC G
  Spe1-f:  ATA ATG AAT GGG GAG GAC TGG TTT ATA AGT TAT TAA TGG G
  Spe1-R:  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.

  prefix: GTTTCTTC GAATTC GCGGCCGC T TCTAG ATGGAAGATAGTTCTTTAGATACTTTAAG
  suffix: GTTTCTTC CTGCAG CGGCCGC  T ACTAGT ACTACTTTAGTAACGGATTAATTTGC       

2. PCR condition

   95°C  30  sec
   95°C  30  sec
   55°C   1  min
   72°C  2.5 min
   72°C  10  min
   cycles:15

3. Ligation to backbones(Psb1C3).

4. Digestion to confirm the fragments

  
  lane 1: 1Kb marker
  lane 2: plasmid(~3Kb)
  lane 3: EcoR1 digestion(~4Kb)
  lane 4: Spe1 digestion(~4Kb)
  lane 5: Pst1 digestion(~4Kb)
  we successfully removed EcoR1 & Spe1 enzyme sites, and added EXSP site at ends of the cry11Aa fragment.

(V) Transform into E.coli

1. Thaw competent cells and BBa_K332011 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.

6. colony PCR to confirm the fragment size (by prefix & suffix primers)

  
  lane 1: 1kb marker
  lane 2: plasmid(cry11Aa+psb1C3)
  lane 3: PCR product(EXSP+cry11Aa) (~2Kb)

7. DNA sequencing:

(VI) Culture with mosquito larvae and observe

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