Part:BBa_K5207020

TS-HydroxySugiol

This composite part consists of six expression cassettes, expressing SmGGPPS, SmCPS1, SmKSL, SmCPR, CYP76AH1, and CYP76AH3.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 4456
  Illegal NheI site found at 11899
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 685
  Illegal BglII site found at 3217
  Illegal BglII site found at 6298
  Illegal BglII site found at 9089
  Illegal BamHI site found at 2815
  Illegal XhoI site found at 2951
  Illegal XhoI site found at 2975
  Illegal XhoI site found at 4268
  Illegal XhoI site found at 4478
  Illegal XhoI site found at 4950
  Illegal XhoI site found at 8131
  Illegal XhoI site found at 11232
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2620
  Illegal NgoMIV site found at 4037
  Illegal NgoMIV site found at 4601
  Illegal NgoMIV site found at 8578
  Illegal NgoMIV site found at 13298
  Illegal AgeI site found at 861
  Illegal AgeI site found at 11195
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 5196
  Illegal BsaI site found at 8117
  Illegal BsaI site found at 8378
  Illegal BsaI.rc site found at 6308
  Illegal BsaI.rc site found at 8367
  Illegal BsaI.rc site found at 9099
  Illegal BsaI.rc site found at 11477
  Illegal SapI.rc site found at 12576
  Illegal SapI.rc site found at 13326
  Illegal SapI.rc site found at 14553
  Illegal SapI.rc site found at 15303
  

Usage and Biology

We combined the Level 1 vectors and constructed them into pYTK096 vector by BsmBI digestion and ligation, and successfully constructed the Level 2 vector.

Figure 1. Snapgene diagrams of pYTK096

Similarly, after transformation in E. coli DH10B identified positive monoclones by resistance, fluorescent labeling screening, and colony PCR.

Figure 2. Fluorescent labeling screening of pYTK095

Figure 3. Validation plot of pYTK096 gel electrophoresis

We can see that the one that does not show green fluorescence is the recombinant plasmid we want, which corresponds to the colony PCR agar gel image, and the recombinant plasmid is successfully constructed. Next, we extracted the corresponding plasmid DNA after amplification and culture, and used it for transformation in yeast cells.

The constructed vector was linearized by NotI endonuclease and then transferred into BY4742 yeast receptor cells, which were screened by ura-deficient medium, and the yeast genomic DNA was extracted for PCR identification to determine the positive single clones.

Figure 4. Graph of the results of the screening of URA-deficient media

Insert fragment PCR assay results:

Figure 5. Gel validation recombinant plasmids in yeast cells

The successful monoclonal plasmid was expanded in culture, shaken small overnight and preserved, and the strain was further fermented and cultured using liquid YPDA medium for 5 days and then centrifuged to collect the organisms.

Figure 6. Schematic diagram of the fermented bacterial liquid

Saccharomyces cerevisiae cells were extracted with methanol by Ultrasonic Cell Disruption for 1 h. After centrifugation at low temperature, the supernatant extract was filtered with a filter membrane, and the filtered samples were transferred to liquid phase vials with lined tubes for Q-Exactive analysis, and the results were as follows:

TY9: as shown in Figure 17, the product of TY9 is mainly composed of two compounds 1,2 which appeared near 6 and 9 minutes, with molecular weights of 317 and 299 under positive mode, respectively. Through literature analysis and discussed with our instructor, we confirmed that the compound MW of 316 is 11-hydroxy-sugiol, and MW 298 has not been found to point to any specific substance at this point in time.

Figure 7. Chromatogram and mass spectrogram of S. c BY4742/TY9 extract.