Team:SDU-Denmark/Tour41
From 2014.igem.org
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.