Part:BBa_K530034

pRSBB415 LEU2 CEN/ARS Yeast Shuttle Vector Yeast shuttle vector for propagation in E. coli and transformation into S. cerevisiae. Contains an Ampicillin resistance marker for E. coli and LEU2 selectable marker for S. cerevisiae. Fully compatible for use with Standard Assembly. Contains EcoRI, XbaI, SpeI, and PstI in the multiple cloning site, in that order. This vector is a CEN/ARS episomal yeast vector.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2799
  Illegal AgeI site found at 1503
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 4479
  Illegal SapI site found at 3396
  Illegal SapI site found at 5500
  

Plasmid Map

Characterization by iGEM uOttawa 2017

iGEM uOttawa 2017 explored if regulated recruitment of dcas9 Mxi1 using synthetic guide RNA is a viable alternative to conventional transcriptional regulatory modules in genetic network engineering.

The main model organism used by our lab and many others is S. Cerevisiae. The absence of available yeast backbones from the Registry and the relatively few available in the Distribution Kit is a significant problem that we sought to investigate. This shuttle vector, BBa_K530034 addresses the issue by enabling the replication and propagation of genetic material in both e coli and yeast.

The existence of this part presented a theoretical solution to bridging the gap between e coli biobricks and the limited compatibility in yeast. However, there is a lack of information regarding its practical usage and application. Therefore, iGEM uOttawa 2017 aimed to improve the characterization of Leu2 CEN/ARS yeast shuttle vector by examining its functionality and applicability to both e coli and yeast systems as a part of our project.

A central part of our project, dcas9 Mxi1, was examined in both e coli and yeast. Our plasmid incorporated the use of Leu2 CEN/ARS yeast shuttle vector that also contained an ampicillin selection (see in plasmid design).

We have proven that this yeast shuttle vector is compatible with ecoli strain DH5alpha as well as yeast strain W303 as shown by the growth on the ampicillin selection plate (ecoli right) and leucine selection plate (yeast left).

In conclusion, our improved informational characterization provides practical support for the use of yeast shuttle vectors as a solution to the long existing compatibility issue.

Contribution Markup

Group: uOttawa, 2017

Author: Sakib Kazi and Yuchen Luo

Summary: We incorporated a plasmid combining Leu2 CEN/ARS yeast shuttle vector and a dcas9 Mxi1 part into the e coli strain DH5alpha and yeast strain w303 and proved its functionality. The success of the experiment provides information regarding practical application of this useful part.

Uploads: For further information regarding the project, please visit http://2017.igem.org/Team:uOttawa Please see images of growth above.