Team:SDU-Denmark/Tour41

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Expressions

Tet construct

We wanted to test if the Tet promoter could be fine-tuned, and what influence the LVA tag on TetR has on the expression.

The ligation of the TetR(+LVA) construct with the pTet-GFP construct was cloned successfully. (Bba_K1475006)

In order for us to ligate the TetR(no LVA) construct with the pTet-GFP construct, we first needed to remove the LVA tag from TetR. The ligation was cloned successfully into a plasmid and can be found in parts registry as Bba_K1475003.

The ligation of TetR(no LVA) construct with the pTet-GFP construct was done successfully and can be found as Bba_K1475005.

Characteriztion/expression

The promoters in the TetR-pTet constructs are supposed to be inhibited by TetR. By induction with doxycycline, the repressor is inhibited, and thus pTet will be active. In this case, GFP will be expressed after induction with doxycycline. Source: Aagaard, L., et al.: A Facile Lentiviral Vector System for Ekspression of Doxycycline-Inducible dhRNAs: Knockdown of the Pre-miRNA Processing Enzyme Drosha. Molecular Therapy, 2007. 15:5, p. 938-945. (Link)

To test if the Tet promoter could be fine-tuned using different concentrations of doxycycline, we ran FACS (Fuorescence-activated Cell Sorting) on E. coli expressing GFP controlled by pTet, regulated by TetR with and without LVA tag. A wild-type was used as control.

Results of the FACS before and after induction with doxycycline.