Coding

Part:BBa_K4182008

Designed by: Dan Tan   Group: iGEM22_XJTU-China   (2022-10-10)
Revision as of 07:15, 12 October 2022 by Dan012 (Talk | contribs)


AA cluster

Synthesis of FPPs, AstA, AatB\AstC gene cluster

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1877
    Illegal EcoRI site found at 4795
    Illegal PstI site found at 126
    Illegal PstI site found at 306
    Illegal PstI site found at 1413
    Illegal PstI site found at 2744
    Illegal PstI site found at 4552
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1877
    Illegal EcoRI site found at 4795
    Illegal PstI site found at 126
    Illegal PstI site found at 306
    Illegal PstI site found at 1413
    Illegal PstI site found at 2744
    Illegal PstI site found at 4552
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1877
    Illegal EcoRI site found at 4795
    Illegal BamHI site found at 184
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1877
    Illegal EcoRI site found at 4795
    Illegal PstI site found at 126
    Illegal PstI site found at 306
    Illegal PstI site found at 1413
    Illegal PstI site found at 2744
    Illegal PstI site found at 4552
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1877
    Illegal EcoRI site found at 4795
    Illegal PstI site found at 126
    Illegal PstI site found at 306
    Illegal PstI site found at 1413
    Illegal PstI site found at 2744
    Illegal PstI site found at 4552
    Illegal NgoMIV site found at 2015
    Illegal NgoMIV site found at 3345
    Illegal AgeI site found at 2170
    Illegal AgeI site found at 3970
  • 1000
    COMPATIBLE WITH RFC[1000]


Profile

Base Pairs

Design Notes

This gene cluster has been optimized for E. coli

Source

LacI:E.coli FPPS:Rhodobacter azotoformans fpps gene for farnesyl diphosphate synthase, partial cds(GenBank: AB053174.1)

Usage&Biology

Construction and validation of AA synthetic plasmid (plasmid 3).

FPPS and astABC (from the soil fungus Aspergillus terreus) were codon-optimized and genetically synthesized according to E. coli, respectively. where astAB and astC are present on two separate plasmids, respectively.

The final plasmid III uses the medium-copy plasmid MCS1 as the backbone (to avoid metabolic stress caused by high-copy plasmids), contains the astABC trigene and specific transcription terminator T1 from the E. coli rrnB gene regulated by the lac promoter, and multiple highly active ribosomal binding sites (RBS1-3). The astABC gene, LacI-Plac regulatory sequence, and MCS plasmid skeleton were obtained using PCR technology, respectively, and the final plasmid 3 was obtained by further one-step ligation using the golden gate technique.

XJTU-p3-4.png

XJTU-p3-5.png

Figure 3: The plasmid in which Ast ABC is located

XJTU-p3-6.png

Figure 4: The MCS skeleton used and a brief illustration

XJTU-p3-7.png

Figure 5: Complete plasmid profile finally constructed by the experimental group

XJTU-p3-8.png

Figure 6: Agarose gel electrophoresis of the LacI gene

XJTU-p3-9.png

Figure 7: Colony PCR glue plot of plasmid 3 (the presence of the marker gene is used to prove the presence of the plasmid)

References

[1]Resistance-gene-directed discovery of a natural-product herbicide with a new mode of action

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