Composite

Part:BBa_K4800008

Designed by: Yifei Wu   Group: iGEM23_NJTech-China-B   (2023-10-06)
Revision as of 18:54, 6 October 2023 by Sariel (Talk | contribs)


Ptrc-sfp-MmCAR(Q302E)-SpyTag-EutM-SpyCatcher-YahK

Ptrc is the promoter, YciA-sfp-Q302E is a mutant gene derived from BBa_K1655000, EutM is the protein scaffold, Yahk is the aldo-keto reductase, and 'molecular glue' SpyTag/SpyCatcher can be used to construct multi-enzyme complexes.


Document

Build:

Fig1. PRSFDuet-YciA-sfp-Q302E-SpyTag-YahK-EutM- SpyCatcher plasmid

The vector was linearized using PCR using PRSFDuet-YciA-sfp-Q302E-YahK as a template. The vector containing the SpyTag sequence was amplified by modifying the 5' end of the primer to amplify an EutM-SpyCatcher-containing fragment from a lab-conserved strain.In-fusion cloning will be used to link the fragment to the linearized vector. The recombinant plasmid will be transfected into the receptor cell Trans-T1 and coated on LB solid medium containing 100 μg/mL kanamycin, and incubated at 37°C for 12 h. Single colonies will be picked for colony PCR validation, and positive colonies will be selected for culture and sequencing to obtain the recombinant plasmid PRSFDuet-YciA-sfp-Q302E-SpyTag-YahK-EutM- SpyCatcher.

Test:

PRSFDuet-YciA-sfp-Q302E-YahK and PRSFDuet-YciA-sfp-Q302E-SpyTag-YahK-EutM-SpyCatcher were respectively transfected into BL21 (DE3) and whole-cell catalyzed, cultured at 30°C 200 rpm for 5 h. Samples were taken at 1-h intervals, and the content of 1,5-PDO was detected by HPLC.

Fig2. Whole-cell catalytic results of KA30 vs KA30 (Q302E+EutM)

By HPLC data, it was seen that BL21 (DE3) imported into PRSFDuet-YciA-sfp-Q302E-SpyTag-YahK-EutM-SpyCatcher catalyzed the production of more 1,5-PDO.

Electrically transfer PRSFDuet-YciA-sfp-Q302E-YahK+PTrc99a-davB-davA-GabT, PRSFDuet-YciA-sfp-Q302E-SpyTag-YahK-EutM-SpyCatcher+PTrc99a-davB-davA-GabT transferred to KA30 ∆YcjQ for fermentation. Samples were taken at 12h intervals for OD600, glucose and lysine content, and the fermentation broth was assayed for 1,5-PDO by HPLC.

Fig3. KA30 and KA30(Q302E+EutM) Fermentation Data

By HPLC data, it was seen that KA30 fermentation imported into PRSFDuet-YciA-sfp-Q302E-SpyTag-YahK-EutM-SpyCatcher produced more 1,5-PDO.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1313
    Illegal EcoRI site found at 5098
    Illegal XbaI site found at 5016
    Illegal SpeI site found at 527
    Illegal PstI site found at 1748
    Illegal PstI site found at 1775
    Illegal PstI site found at 3044
    Illegal PstI site found at 3071
    Illegal PstI site found at 3464
    Illegal PstI site found at 3960
    Illegal PstI site found at 4604
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1313
    Illegal EcoRI site found at 5098
    Illegal NheI site found at 5786
    Illegal SpeI site found at 527
    Illegal PstI site found at 1748
    Illegal PstI site found at 1775
    Illegal PstI site found at 3044
    Illegal PstI site found at 3071
    Illegal PstI site found at 3464
    Illegal PstI site found at 3960
    Illegal PstI site found at 4604
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1313
    Illegal EcoRI site found at 5098
    Illegal BglII site found at 2302
    Illegal BglII site found at 2928
    Illegal BglII site found at 3204
    Illegal BglII site found at 3895
    Illegal BamHI site found at 1285
    Illegal BamHI site found at 4859
    Illegal BamHI site found at 5092
    Illegal XhoI site found at 4844
    Illegal XhoI site found at 4931
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1313
    Illegal EcoRI site found at 5098
    Illegal XbaI site found at 5016
    Illegal SpeI site found at 527
    Illegal PstI site found at 1748
    Illegal PstI site found at 1775
    Illegal PstI site found at 3044
    Illegal PstI site found at 3071
    Illegal PstI site found at 3464
    Illegal PstI site found at 3960
    Illegal PstI site found at 4604
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1313
    Illegal EcoRI site found at 5098
    Illegal XbaI site found at 5016
    Illegal SpeI site found at 527
    Illegal PstI site found at 1748
    Illegal PstI site found at 1775
    Illegal PstI site found at 3044
    Illegal PstI site found at 3071
    Illegal PstI site found at 3464
    Illegal PstI site found at 3960
    Illegal PstI site found at 4604
    Illegal NgoMIV site found at 3572
    Illegal NgoMIV site found at 3634
    Illegal NgoMIV site found at 3851
    Illegal NgoMIV site found at 4298
    Illegal AgeI site found at 1478
    Illegal AgeI site found at 2489
    Illegal AgeI site found at 2786
    Illegal AgeI site found at 3671
    Illegal AgeI site found at 3680
    Illegal AgeI site found at 4355
  • 1000
    COMPATIBLE WITH RFC[1000]


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