Part:BBa_K3996012

pXynA

pXynA

Profile

Properties: A Yeast Expression plasmids backbone

Usage and Biology

It is formed with pOdd-3 plasmid backbone, FBA1 promoter, AnXlnD orf and CYC1 terminator.

Construct design

The plasmid is engineered for further use. (Figure 1).

Figure 1. DNA map of plasmid pXylan..

Experimental approach

1. Fragments PCR products Electrophoresis To utilize the xylan component contained in the wheat B starch, we cloned the xylanase expression gene from Aspergillus niger.

Figure 2. Plasmids construction used fragments PCR amplification..

(A) Lane 1: GAP promoter, 695 bp. Lane 2: AnXlnB CDS, 706 bp. Lane 3: CYC1 terminator, 276bp. Lane 4: pXlnB plasmid backbone fragment, 1757 bp. Lane 5: TPI1 promoter, 614 bp. Lane 6: AnXlnD CDS, 2443 bp. Lane 7: pXlnD plasmid backbone fragment, 1804 bp. (B) Lane 1: pXlnB plasmid backbone fragment, 1804 bp. Lane 2: pXlnD plasmid backbone fragment, 1804 bp. For the pXlnB plasmid construction, the promoter GAP, codon-optimized AnXlnB CDS, and CYC1 terminator PCR bands were shown in the Figure 3A, lane 1, lane2, and lane 3, respectively. The AnXlnB expression cassette was obtained through the overlap PCR. The backbone fragment (kanR with ori) was amplified using two round PCR, the first round and the final fragment band were shown in Figure 3A lane 4 and Figure 3B lane 1, respectively. The backbone was cut with Bsa1 restriction enzyme and ligated with the AnXlnB expression cassette to make the plasmid pXlnB.

References

3. Claes A, Deparis Q, Foulquié-Moreno M R, et al. Simultaneous secretion of seven lignocellulolytic enzymes by an industrial second-generation yeast strain enables efficient ethanol production from multiple polymeric substrates[J]. Metabolic engineering, 2020, 59: 131-141.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 885
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 885
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1278
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 885
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 885
  Illegal NgoMIV site found at 2062
  Illegal AgeI site found at 1655
  
• 1000
  COMPATIBLE WITH RFC[1000]