Part:BBa_K4188001

pSB3CY_aeBlue shuttle vector backbone for Saccharomyces cerevisiae

This Shuttle vector is a further development of the iGEM standard vector pSB3C01. This development allows it to be used as an expression vector for Saccharomyces cerevisiae without the presence of a selection marker in the multi-transcription unit. For this purpose, the 2μ Ori (BBa_K1430000), the aeBlue chromoprotein (BBa_K864401) flanked by the promoter (BBa_J23110) and the terminator (BBa_B0015) were added to the vector (pSB3C01). The new chromoprotein cassette can be excised via a Golden Gate Assembly with the enzyme BsmBI and exchanged for the desired selection marker. This combination of two chromoproteins can be seen during cultivation by a color reaction which shows the modifications. The initial vector shows a purple color, if an auxotrophy marker is now added via Golden Gate Assembly, the successful transformants turn red. Conversely, a successful Golden Gate Assembly with a multi-transcription unit shows blue staining of the colonies. If both chromoproteins are exchanged, the transformants become white and the vector can be transformed into Saccharomyces cerevisiae after plasmid extraction. If the selection marker needs to be changed after a Golden Gate Assembly has been performed, this can be done retrospectively using the enzymes NotI, SpeI, and BamHI.

Ready to use vectors

Part number	Selection marker
BBa_K4188002	Histedine
BBa_K4188003	Leucine
BBa_K4188004	Tryptophane
BBa_K4188005	Uracile

Color characterisation

The newly designed level 2 shuttle vector can be characterized by the color shown after transformation. While the initial vector shows a purple color, successful transformants turn red if an auxotrophy marker is added via Golden Gate. Furthermore, the insertion of a successful Golden Gate Assembly with a multi-transcription unit into the site of the red chromoprotein (mRFP1) gene the vector shows blue staining of the colonies. If both chromoproteins are exchanged, the transformants become white and can be transformed into S. cerevisiae after plasmid extraction.

Figure 1: left picture: Escherichia coli colonies transformed with BBa_K4188001 middle picture: blue E. coli colonies after insertion of an multi transcription unit
right picture: red E. coli colonies after insertion of a Saccharomyces cerevisiae selection marker.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 6155
  Illegal XbaI site found at 3425
  Illegal SpeI site found at 3790
  Illegal PstI site found at 1105
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 6155
  Illegal NheI site found at 3849
  Illegal NheI site found at 3872
  Illegal NheI site found at 4841
  Illegal SpeI site found at 3790
  Illegal PstI site found at 1105
  Illegal NotI site found at 4782
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 6155
  Illegal BamHI site found at 2441
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 6155
  Illegal XbaI site found at 3425
  Illegal SpeI site found at 3790
  Illegal PstI site found at 1105
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 6155
  Illegal XbaI site found at 3425
  Illegal SpeI site found at 3790
  Illegal PstI site found at 1105
  Illegal AgeI site found at 793
  Illegal AgeI site found at 905
  Illegal AgeI site found at 2083
  Illegal AgeI site found at 2406
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 1378
  Illegal BsaI site found at 6161
  Illegal BsaI.rc site found at 1099
  Illegal SapI site found at 1082
  Illegal SapI.rc site found at 6