Difference between revisions of "Part:BBa K4800009"
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<p style="font-size: 160%; font-weight: bold;">1.Experimental Methods | <p style="font-size: 160%; font-weight: bold;">1.Experimental Methods | ||
| − | <p>1.1 Plasmid construction | + | <p>1.1 Plasmid construction<br> |
Firstly, the laboratory conserved strain was used as a template, davB and davA and homologous arms were amplified by PCR, and then the davB-davA fragment was obtained by overlap PCR to obtain davB-davA fragment, the above PCR amplified davB-davA fragment gene and linearized pTrc99a body were connected by one-step cloning, transferred into the receptor cell Trans-T1 and coated in LB solid medium containing 100 μg/mL ampicillin for cultivation, and single colonies on the plate were selected for verification, and the positive colonies were transfected into The positive colonies were transferred to 5mL LB medium at 37℃, 200rpm for incubation. 10h later, the plasmid was extracted for sequencing and validation, and the recombinant plasmid PTrc99a-davB-davA was obtained. the existing strains in the laboratory were used as templates, and GabT and the homology arm were amplified by PCR, and the fragment GabT was linearized by the enzymatic cleavage of the fragment GabT with Sac Ⅰ and Xba Ⅰ by the In-fusion cloning method. The fragment GabT was homologously recombined with SacⅠand XbaⅠenzymatically linearized vector PTrc99a-davB-davA by In-fusion cloning to obtain PTrc99a-davB-davA-GabT. | Firstly, the laboratory conserved strain was used as a template, davB and davA and homologous arms were amplified by PCR, and then the davB-davA fragment was obtained by overlap PCR to obtain davB-davA fragment, the above PCR amplified davB-davA fragment gene and linearized pTrc99a body were connected by one-step cloning, transferred into the receptor cell Trans-T1 and coated in LB solid medium containing 100 μg/mL ampicillin for cultivation, and single colonies on the plate were selected for verification, and the positive colonies were transfected into The positive colonies were transferred to 5mL LB medium at 37℃, 200rpm for incubation. 10h later, the plasmid was extracted for sequencing and validation, and the recombinant plasmid PTrc99a-davB-davA was obtained. the existing strains in the laboratory were used as templates, and GabT and the homology arm were amplified by PCR, and the fragment GabT was linearized by the enzymatic cleavage of the fragment GabT with Sac Ⅰ and Xba Ⅰ by the In-fusion cloning method. The fragment GabT was homologously recombined with SacⅠand XbaⅠenzymatically linearized vector PTrc99a-davB-davA by In-fusion cloning to obtain PTrc99a-davB-davA-GabT. | ||
| − | 1.2 Fermentation | + | <br>1.2 Fermentation<br> |
PTrc99a-davB-davA-GabT and PCWJ-YciA-sfp-MmCAR-YahK were transferred into the KA30 sensory state of the original strain by electrotransfer, coated on LB solid medium containing 50 μg/mL ampicillin and 34 μg/mL chloramphenicol, and incubated at 37℃ for 48 h. Single colonies were selected and colony PCR was performed. The PCR bands were analyzed by agarose gel electrophoresis, and the positive colonies were selected and inoculated into 5 mL of KA30 seed medium and cultured at 37℃ for 20 hours. Take 2mL of seed liquid and inoculate it into KA30 fermentation medium. Put in 37℃ 200rpm culture until OD600 was 0.6, add 50mM IPTG and 0.55g/L α-ketoglutaric acid, put in 30℃ 200rpm culture for 72 hours, take samples every 12h to test the content of OD600, glucose and lysine, and detect the content of 1,5-PDO in the fermentation broth by HPLC. | PTrc99a-davB-davA-GabT and PCWJ-YciA-sfp-MmCAR-YahK were transferred into the KA30 sensory state of the original strain by electrotransfer, coated on LB solid medium containing 50 μg/mL ampicillin and 34 μg/mL chloramphenicol, and incubated at 37℃ for 48 h. Single colonies were selected and colony PCR was performed. The PCR bands were analyzed by agarose gel electrophoresis, and the positive colonies were selected and inoculated into 5 mL of KA30 seed medium and cultured at 37℃ for 20 hours. Take 2mL of seed liquid and inoculate it into KA30 fermentation medium. Put in 37℃ 200rpm culture until OD600 was 0.6, add 50mM IPTG and 0.55g/L α-ketoglutaric acid, put in 30℃ 200rpm culture for 72 hours, take samples every 12h to test the content of OD600, glucose and lysine, and detect the content of 1,5-PDO in the fermentation broth by HPLC. | ||
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Revision as of 16:18, 6 October 2023
davB-davA-GabT
The fragment expresses L-lysine monooxygenase, 5-aminopentanamidase, and 4-aminobutyrate aminotransferase GabT, respectively, which catalyze the reactions: L-lysine + dioxygen → 5-aminopentanamide + CO2 + H2O, 5-aminopentanamide + H2O → 5-aminopentanoate + ammonium 5-aminopentanoate + 2-oxoglutarate ↔ L-glutamate + glutarate semialdehyde Constitutes the Lysine to 5-oxopentanoate pathway
Build:
1.Experimental Methods
1.1 Plasmid construction
Firstly, the laboratory conserved strain was used as a template, davB and davA and homologous arms were amplified by PCR, and then the davB-davA fragment was obtained by overlap PCR to obtain davB-davA fragment, the above PCR amplified davB-davA fragment gene and linearized pTrc99a body were connected by one-step cloning, transferred into the receptor cell Trans-T1 and coated in LB solid medium containing 100 μg/mL ampicillin for cultivation, and single colonies on the plate were selected for verification, and the positive colonies were transfected into The positive colonies were transferred to 5mL LB medium at 37℃, 200rpm for incubation. 10h later, the plasmid was extracted for sequencing and validation, and the recombinant plasmid PTrc99a-davB-davA was obtained. the existing strains in the laboratory were used as templates, and GabT and the homology arm were amplified by PCR, and the fragment GabT was linearized by the enzymatic cleavage of the fragment GabT with Sac Ⅰ and Xba Ⅰ by the In-fusion cloning method. The fragment GabT was homologously recombined with SacⅠand XbaⅠenzymatically linearized vector PTrc99a-davB-davA by In-fusion cloning to obtain PTrc99a-davB-davA-GabT.
1.2 Fermentation
PTrc99a-davB-davA-GabT and PCWJ-YciA-sfp-MmCAR-YahK were transferred into the KA30 sensory state of the original strain by electrotransfer, coated on LB solid medium containing 50 μg/mL ampicillin and 34 μg/mL chloramphenicol, and incubated at 37℃ for 48 h. Single colonies were selected and colony PCR was performed. The PCR bands were analyzed by agarose gel electrophoresis, and the positive colonies were selected and inoculated into 5 mL of KA30 seed medium and cultured at 37℃ for 20 hours. Take 2mL of seed liquid and inoculate it into KA30 fermentation medium. Put in 37℃ 200rpm culture until OD600 was 0.6, add 50mM IPTG and 0.55g/L α-ketoglutaric acid, put in 30℃ 200rpm culture for 72 hours, take samples every 12h to test the content of OD600, glucose and lysine, and detect the content of 1,5-PDO in the fermentation broth by HPLC.
Test:
Result
The HPLC data showed that the 72h fermentation broth contained 62 mM of 1,5-pentanediol, which indicated that the gene pathway we designed and constructed was viable
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 269
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1358
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1518
Illegal AgeI site found at 925
Illegal AgeI site found at 1174
Illegal AgeI site found at 2896 - 1000COMPATIBLE WITH RFC[1000]