Part:BBa_K5508008

Gal1,10-HsLF-mCherry-CYC1

We combined BBa_K4335004 with BBa_K5508001 to verify whether lactoferrin was successfully expressed.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1833
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1833
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2248
  Illegal BglII site found at 2372
  Illegal BglII site found at 3359
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1833
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1833
  Illegal NgoMIV site found at 3215
  Illegal AgeI site found at 1247
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 2712
  

Results

(1)Plasmid Construction

The target gene mCherry was amplified with HsLF-mCherry-F（CACCATCACCACCACTAATAAAAGCTTatggtgagcaagggcgaggag） and HsLF-mCherry-R（GGATCTTAGCTAGCCGCGGTACCttacttgtacagctcgtccatg）, and the BBa_K5508000 vector was digested with Hind3 and Kpn1, followed by Gibson assembly.

Electrophoretic detection of PCR results

Results of E. coli plasmid transformation

Sequencing results

(2)Fluorescence detection

After the correctness of the recombinant plasmid was verified, it was transferred into BY4741 for culture. After 48 hours of adding the inducer galactose, the results are shown.

Results of plasmid transformation in Saccharomyces cerevisiae

Expression of fluorescent protein：The figure shows from left to right: negative control, negative control, pESC-mCherry, pESC-HsLF-mCherry

It can be seen that after induction, the strain shows a pink colour when illuminated by UV light. However, this colour can only be seen after centrifugation of the collected bacteria and cannot be observed directly by the naked eye in the liquid. In particular, pESC-HsLF-mCherry/BY4741 was lighter in colour compared to pESC-mCherry/BY4741. This may be due to the expression of HsLF in Saccharomyces cerevisiae low.