Part:BBa_K4182008:Design

AA cluster

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

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.

Figure 3: The plasmid in which Ast ABC is located

Figure 4: The MCS skeleton used and a brief illustration

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

Figure 6: Agarose gel electrophoresis of the LacI gene

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