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.
The results of the FACS illustrates that without induction with doxycycline, GFP is still expressed. This is
because the promoter is leaky. There is a very little variation in expression of GFP upon induction with low
concentration of doxycycline. At high concentration of doxycycline (2000 ng/mL) it can clearly be seen that
TetR (+LVA) is more inhibited than TetR without LVA.
Leaving the leakiness of pTet out of account, FACS results indicates that the pTet inhibited by TetR with LVA
tag is the one most active, upon induction by doxycycline.
Because pTet is leaky, all cells express GFP. It can be difficult to tell if the pTet has been induced and to
what extent, however, plates containing the corresponding concentrations of doxycycline as used in FACS
clearly shows an induction.
Duplicates of plates with doxycycline were made with 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500
ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline. On the plates, TetR-pTet construct with LVA, TetR-pTet
construct with no LVA, pTet-GFP without TetR construct and wild-type were plated.