Part:BBa_K2623015

Secretory-abundant heat soluble protein "SAHS " (promoter, RBS, RFP and double terminator)

TDP circuit is used to express exocrine protein SAHS. In order to facilitate the characterization and purification of SAHS protein, we added the promoter J23100+RBS+SAHS protein+RBS+E1010+B0015 to pSB1C3. TDP circuit is used to express exocrine protein SAHS BBa_K2623007. We added RFP BBa_E1010 as a reporter gene to the gene circle for us to screen and validate. Because our aim is to obtain a large number of SAHS proteins, the constant promoter J23100 is chosen to allow SAHS to be expressed continuously. The sequence encoding for the gene is preceded by a constant promoter BBa_J23100 and an RBS BBa_B0034 , and followed by a double terminator BBa_B0015.

In our circuit of the build process, we have been doing nucleic acid gel electrophoresis to verify. After the loop is complete, sequencing verification.




After the fluorescence identification, we performed a small amount of protein expression by SDS-PAGE.


SAHS is an exogenous protein with a signal peptide in front of it that allows the protein to be secreted outside the membrane. But our site organism is E. coli, because there is a cell wall, so the protein can not be secreted out of the membrane. Therefore, we can hardly see our target protein in the supernatant. Therefore, we can only obtain the protein we need from the cell pellet. After that, we connected our SAHS protein to the pET-28a plasmid, induced expression, and obtained a large amount of our target protein by ultrasonic disruption of the bacterial cell pellet.
More information about our project can be found on our results page.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 279
  Illegal XhoI site found at 503
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1152
  Illegal AgeI site found at 1264
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 558
  Illegal SapI.rc site found at 553