Part:BBa_K3930013

Down integrative sequence to target locus X-3 of S. cerevisiae genome Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 71
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 71
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 71
  Illegal XhoI site found at 378
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 71
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 71
  
• 1000
  COMPATIBLE WITH RFC[1000]

Introduction

Integrative locus down X-3 of S. cerevisiae genome comes from the EasyClone-MarkerFree kit (Jessop-Fabre et al.,2016). This part needs to flank the insertion in 3', and must be used with the integrative locus (BBa_K3930012) X-3 up part in 5'.

Results

Integration of the part (BBa_K3930001) into the yeast genome

The part (BBa_K3930001) was linearized and transformed into the S.cerevisiae LycoYeast strain according to the Takara yeast transformation protocol, with 5 µg of DNA. The construction is flanked by the part (BBa_K39300012) and (BBa_K39300013). Figure 1 shows the electrophoresis gel of PCR on colony to verify clones.

Figure 1: Integration of pFRAMBOISE in the LycoYeast genome

pFRAMBOISE was checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented on the right of each gel and the NEB 1 kb DNA ladder on the left (note that a different ladder is presented on the theoretical gel)

The Integrative locus X-3 down (BBa_K3930013) coupled with the integrative locus (BBa_K3930012) X-3 up part work under those experimental conditions.

References

1. Chen X, Shukal S, Zhang C. 2019. Integrating Enzyme and Metabolic Engineering Tools for Enhanced α-Ionone Production. J Agric Food Chem. 67(49):13451–13459. doi:10.1021/acs.jafc.9b00860.