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]Illegal BamHI site found at 1427
Illegal BamHI site found at 1766
Illegal XhoI site found at 547 - 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
Results of Agarose Gel Electrophoresis
After the electro-transformed colonies were cultured, a small amount of bacterial liquid was taken for colony PCR. PCR primers are selected from upstream and downstream sequences outside the component.
The experimental results are as follows. It can be clearly seen that there are bands with the correct length in the PCR products, indicating that this part has been integrated into the A.caul genome.
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