Part:BBa_K4937041

TPI1p-SPE3-PRM9t-TEF1p-AtACL5-CYC1t-TDH3p-SPE2-DIT1t

This part belongs to our plasmid library, and the library is used to stably express SPE2 and tune the expression level of SPE3 and AtACL5 to fully use the spermidine substrate and examine the numerical relationship between spermidine and thermospermine.

We firstly achieved all short single fragment by PCR from the genome of S. cerevisiae and plasmid (Figure 1). Then we connected all single expression device by fusion PCR (Figure 2).

Figure 1

Figure 2

However, the Gibson coloning results were not as expected. We performed the double enzyme digestion experiment and PCR to check whether the sequence of whole plasmids are right. And only one colony shows expected results(Figure 3), we guessed it may come from the unexpected similarity of homologous fragment. Due to time limited, we didn’t complete this part of work. However, we will keep on trying this design.

Figure 3

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1516
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 3421
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1881