Team:SDU-Denmark/Tour41

Expressions

Characerization of TetR/pTet

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 and is found as 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 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 cloned 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.

Figure 1: 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) inhibits pTet at a weaker extent than TetR without LVA.

To analyse the amount of TetR with and without LVA tag present in the cell, coommassie stainging was made on a SDS-page with E. coli K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.

Figure 2: Coomassie staining on with E. coli K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively. The coomassie staining shows that the construct expressing TetR(+LVA) expresses more GFP than the construct expressing TetR(no LVA). In addition to this, the staining shows a higher amount of TetR(no LVA) in the cell than of TetR(+LVA). This is consistent with the FACS results that illustrates that pTet-TetR(+LVA) expresses more GFP than pTet-TetR(no LVA). The coomassie staining indicates that the reason for the higher expression of GFP by pTet-TetR (+ LVA) is because the cell contains less inhibitor. This must be due to the LVA tag making TetR unstable and tagging it for degredation.

Leaving the leakiness of pTet out of account, FACS results indicate that the pTet inhibited by TetR with LVA tag is the one most active, upon induction by doxycycline. This could be because the LVA tag makes TetR more unstable and tags it for degredation or because TetR with LVA is better inhibited by doxycycline than TetR without LVA.

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

	0 ng/mL doxycycline	50 ng/mL doxycycline	100 ng/mL doxycycline
Second 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

The plating of TetR-GFP constructs on plates with doxycycline shows that GFP is expressed at different levels at different concentrations of doxycycline. Expression increases with an increase in doxycycline concentrations. The plates also show that GFP, to some extent, is expressed without doxycycline. This indicates that the Tet promoter is leaky and is not fully inhibited by TetR as it could be seen in the FACS results.

To see how the growth of the bacteria expressing GFP controlled by pTet are affected, we have measured OD over 8 hours. We measured OD on triplicates of bacteria with an empty vector, pTet-GFP, pTet (no LVA)- GFP, pTet (+LVA)-GFP and a wild-type.

Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.

Figure 3 shows the growth of bacteria expressing GFP constitutiely, are attenuated the most with most comprised growth. Removing the LVA tag from TetR also has a negative effect on the growth of the bacteria. This could be because TetR without LVA stresses the metabolism of the bacteria more than TetR with LVA or because LVA tags TetR for degradation and thus TetR with LVA stresses the cell less than TetR without LVA.

Characterization of lacI/plac

2013 SDU-Denmark iGEM team proved that the natural lac inhibitor has a faster respondance on induction by IPTG, than lacI with LVA (Link). As for pTet, we wanted to test if the lac promoter could be fine-tuned. Due to the 2013 SDU iGEM team, we used the lacI without LVA. We wanted to ligate a constitutive promoter-lacI (no LVA) with plac-GFP. This was done successfully and can be found as Bba_K1475007. Due to time constrains, we were never able to characterize this part and compare it to pTet.