Difference between revisions of "Part:BBa K4115041"
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<partinfo>BBa_K4115041 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4115041 SequenceAndFeatures</partinfo>
 
==Related Experiments==
 
==Related Experiments==
−
 
+
===Preparation of Competent A.caul Cells===
 +
- Remove the glycerol-preserved ORS571 from -80°C, transfer it to 5ml of Amp TY medium, and culture at 37°C on a shaker for 2 days<br>
 +
- 1:100 was inoculated into 100ml of Amp TY medium, cultured at 37°C with OD = 0.4-0.6, the culture was placed on ice for 30min, aliquoted into 50ml EP tubes, centrifuged at 6000rpm at 4°C for 10min, and the supernatant was discarded<br>
 +
- 35ml of sterile pre-cold water to suspend bacteria<br>
 +
- Centrifuge at 6000rpm at 4°C for 10min to discard the supernatant, then use 20ml of sterile pre-cooled water to suspend the cells<br>
 +
- repeat<br>
 +
- Use 2.5ml of sterile cold 10% glycerol to suspend the bacterial cells, dispense 150uL per tube into 1.5ml EP tubes, and store at -80°C for later use<br>
 
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<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K4115041 parameters</partinfo>
 
<partinfo>BBa_K4115041 parameters</partinfo>
 
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Revision as of 11:19, 4 October 2022


nifA_upstream-BleoR-nifA_downstream


Usage and Biology

Add the upstream and downstream fragments of the nitrogen-fixing bacteria (A. caul) nifA gene upstream and downstream of the bleomycin resistance gene. This component was used in the project to edit the nitrogen-fixing bacteria (A. caul) genome by genetic recombination methods.[1]
This part can also be used to mark and screen colonies with successful gene knockout, and to prove whether homologous recombination method can effectively edit the A. caul genome.
[1]Ryu, M. H., Zhang, J., Toth, T., Khokhani, D., Geddes, B. A., Mus, F., Garcia-Costas, A., Peters, J. W., Poole, P. S., Ané, J. M., & Voigt, C. A. (2020). Control of nitrogen fixation in bacteria that associate with cereals. Nature microbiology, 5(2), 314–330. https://doi.org/10.1038/s41564-019-0631-2

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Unknown
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 434
  • 21
    INCOMPATIBLE WITH RFC[21]
    Unknown
  • 23
    INCOMPATIBLE WITH RFC[23]
    Unknown
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 234
    Illegal NgoMIV site found at 339
    Illegal NgoMIV site found at 556
    Illegal NgoMIV site found at 1179
    Illegal NgoMIV site found at 1240
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 331
    Illegal BsaI.rc site found at 1287

Related Experiments

Preparation of Competent A.caul Cells

- Remove the glycerol-preserved ORS571 from -80°C, transfer it to 5ml of Amp TY medium, and culture at 37°C on a shaker for 2 days
- 1:100 was inoculated into 100ml of Amp TY medium, cultured at 37°C with OD = 0.4-0.6, the culture was placed on ice for 30min, aliquoted into 50ml EP tubes, centrifuged at 6000rpm at 4°C for 10min, and the supernatant was discarded
- 35ml of sterile pre-cold water to suspend bacteria
- Centrifuge at 6000rpm at 4°C for 10min to discard the supernatant, then use 20ml of sterile pre-cooled water to suspend the cells
- repeat
- Use 2.5ml of sterile cold 10% glycerol to suspend the bacterial cells, dispense 150uL per tube into 1.5ml EP tubes, and store at -80°C for later use