Part:BBa_K814015

G418 selection yeast shuttle vector designed for BioBrick

A yeast/E. coli shuttle vector was designed with BioBrick prefix and suffix sites such that a yeast expression cassette could be inserted through basic BioBrick assembly. G418 was chosen instead of an auxotrophic marker for plasmid selection in yeast.

To synthesize the overall plasmid backbone, PCR primers were designed to amplify five desired fragments (with 25-30 bp overlap) from different plasmids, which could be joined by Gibson Assembly: 1. BioBrick destination plasmid pSB1C3 containing the MCS and rep (pMB1). 2. BioBrick destination plasmid pSB1C3 containing the Chloramphenical resistance gene. 3. 2u Gibson Extract provided by the Schmidt-Dannert lab (University of Minnesota) containing the 2u ORI for yeast. 4. pkT127 provided by the Schmidt-Dannert lab containing G418 resistance genes and tTEF1. 5. ATCC plasmid provided by the Schmidt-Dannert lab containing the ADH1 promoter to drive the G418 R gene.

Figure 1: Pieces for Gibson assembly of our novel, shuttle backbone. The individual pieces relate to the described components enumerated above. 1. Purple arrow; 2. Orange arrow; 3. Green arrow; 4. Blue arrow; 5. Light green arrow

Figure 2: A novel BioBrick shuttle vector. Gibson assembly was used to fuse components of the pSB1C3 shipping vector to replication and selection machinery for S. cerevisiae. A BioBrick MCS was maintained to allow for modular design of synthetic circuits destined for expression in bacterial or yeast-based systems.

Figure 3. PCR screening of caffeine biosynthetic genes from yeast . Construct maps synthesized from G-Blocks (IDT) are shown below.

Sequence and Features

Assembly Compatibility:

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

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 5682
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 5688
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 5682
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 5682
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 5682
  Plasmid lacks a suffix.
  Illegal XbaI site found at 5697
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NgoMIV site found at 5355
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI.rc site found at 4705