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.

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.

0 ng/mL doxycycline 50 ng/mL doxycycline 100 ng/mL doxycycline
First series of plating of TetR-GFP at different concentrations of doxycycline






200 ng/mL doxycycline 500 ng/mL doxycycline 1000 ng/mL doxycycline 2000 ng/mL doxycycline