Part:BBa_K3369000:Design

B.subtilis Nitric Oxide synthase

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 45
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 45
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 45
  Illegal BglII site found at 200
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 45
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 45
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 295
  

Design Notes

This part is intergrated into pET-28a plasmid to test the function. Still, silent mutation is needed to be made to meet the needs of assembly compability.

Bacterial strains and Culture The Bacillus Subtilis used for NOS gene source is wild type Bacillus Subtilis 168 from Pro Chen Guoqiang. The E.coli used for plasmid amplification is DH5α from Tiangen (China). The E. coli used for NOS protein expression is BL21 (DE3) from Tiangen (China). All the strains are cultured at the LB. The transformed bacterial will be cultured at LB with 100μg/ml kanamycin at 37℃

DNA Digests We get the PET28-a from former Tsinghua iGEM team and restriction enzyme from BioLabs (New England). The DNA digests is operation according to the following system in a PCR tube. DNA sample 1μg NEB 3.1buffer 2μl BamHI 1μl NcoI 1μl Add ddH2O to final volume 20μl Incubate the mixture at 37℃ for 1h.

IPTG induce Monoclones were selected and added with 2mlLB (containing 100μg/ml kanamycin). Concussion culture at 37℃ overnight. Arginine was added to 40ml LB(containing 100μg/ml kanamycin) to final 0.5mM concentration. ALA was added to final 0.45mM concentration and 400μl microbial was added. Then Incubate at 37℃ until OD600=0.5. Absorb half of the liquid and add it to the new conical flask as control. IPTG was added to the induced group to final 0.5mM concentration. Then incubate at 28℃.

No Detection We buy the Total No detection kit from Beyotime (China) After the addition of IPTG for 0,1,2,3,4 and 5 hours, 1ml liquid was taken out respectively. Centrifuge at 12000rpm for 1min. Take 60μl of supernatant and add it to the 96-well plate. Repeat for three times. We diluted 1M sodium nitrite with LB to 0μM ,1μM ,2μM,5μM ,10μM ,20μM,40μM,60μM,80μM to make a standard solution Then we added 5μl 2mM NADPH, 10μl FAD, 5μl Nitrate Reductase in each hole. We incubated the plate at 37℃ for 30min. We added 10μl LDH buffer, 10μl LDH in each hole. We incubated the plate at 37℃ for 30min. Finally, we added 50μl Griess Reagent I, 50μl Griess Reagent II in each hole and detected OD540 using enzyme standard instrument

Western blot After the addition of IPTG for 0,1,2,3,4 and 5 hours, 1ml liquid was taken out respectively. Centrifuge at 12000rpm for 1min. The mass of the bacteria was measured. For 1mg, added 1μl of 2Xloading buffer and suspension the bacteria. Then heat the solution at 100℃ for 10min. The sample was loaded at the 4% stock gel and 12.5%% separation SDS-PAGE gel. Run the electrophoresis at 140V for 20 min, then changed voltage to 160V for 80 min. Proteins were transferred to NC membrane by wet transfer membrane method at 300mV, 2h. After that, the NC membrane was cut between the 25-48KD. The cut membrane was incubated with 10ml 8% milk (TBST dissolved) at 37℃ for 1h. Then the 1mg/ml His-tag Mouse Monoclonal Antibody(YTHX) was diluted in 8% milk (TBST dissolved). The NC membrane was added to the 10ml diluted first antibody at 4℃ for overnight. Then the NC membrane was washed with TBST at room temperature for 10min for three times. The 100μg/ml Goat Anti-mouse IgG(H&L)-HRP Conjugated (EASYBIO) was diluted in TBST. The NC membrane was added to the 10ml diluted second antibody at 37℃ for 1h. Then the NC membrane was washed with TBST at room temperature for 10min for three times. The protein signal was obverse with Western Lighting ECL Pro (Tamper Evident seal)

Source

Cloned from B.subtilis genome.

References

Liu P , Huang Q , Chen W . Heterologous expression of bacterial nitric oxide synthase gene: a potential biological method to control biofilm development in the environment[J]. Canadian Journal of Microbiology, 2012, 58(3):336.