Team:TU Munich/Project/Vector Design

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Contents

Vector Design


Responsible: Ingmar Polte

Motivating text why we choose pYES2 and why we wanted to introduce a new vector to the registry.

Design


vom Poster:

RFC25 COMPATIBILITY

To be able to test and quantify the expression of desired enzymes in yeast we designed an expression vector which is compatible to the iGEM RFC25 standard based on the commercially available pYES2 vector from Invitrogen.

DESIGN

This included an exchange of the original multiple cloning site by a multiple cloning site consisting of the restriction enzymes used in the iGEM RFC25 standard. Additionally the restriction sites of these enzymes in the vector backbone have been eliminated by quick-change mutagenisis. Furthermore we inserted a gene sequence coding for a strep-tag II straight behind the multiple cloning site in order to facilitate the extraction of expressed proteins. The successful introduction of these modifications has been confirmed via DNA sequencing. Additionally a second vector containing a His-tag instead of the strep-tag II and a vector giving the opportunity to test different promoters is currently planned.


Description of the original vector + 4 different modified versions.

Results


BioBricks

Gel Pictures of finished constructs

Characterization

Induction Assays with Galactose

Characterization of Constitutive Promoters

Reference