Part:BBa_K4115041
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 434
- 21INCOMPATIBLE WITH RFC[21]Unknown
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000INCOMPATIBLE 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
Shock Transformation
- Wash the 0.1cm rotor cup with sterile water, 75% ethanol and absolute ethanol, dry it on a clean bench, and place it in -20°C ethanol
- Thaw a few ORS571 electro-competent cells on ice and add 6ul of recombinant plasmid (including BBa_K4115041). After ice bath for 30min, transfer to the rotor cup while avoiding the generation of air bubbles in the rotor cup
- Electroporator, 2.5kV 5ms electroporation, add 1ml TY medium to the ultra-clean bench, transfer to 1.5ml EP at 37°C 180r/min for recovery
- Centrifuge at 8000rpm for 1min after 7h, discard 900uL of supernatant, resuspend and coat on resistant YMA solid medium, and culture at 37°C
| None |