Team:SDU-Denmark/Tour41

Expressions

Characerization of TetR/pTet

As mentioned on the System design page, we wanted to investigate the Tet promoter expression profile, and what influence the LVA tag on the pTet promoter regulator (TetR) had on the expression profile.

For this purpose three plasmids were constructed. The first expressing GFP from pTet promoter with no regulation. The second and third constitutively expressing TetR with or without the LVA tag as well

The pTet-GFP construct was cloned.

A TetR (no LVA) construct was cloned by PCR amplification without the LVA tag and addition of promoter, RBS and terminator. Subsequently the construct was ligated into the pTet-GFP construct. The constructs can be found in parts registry as Bba_K1475004 and Bba_K1475005, respectively.

A TetR (with LVA) construct was cloned by PCR amplification with the LVA tag and addition of the same promoter, RBS and terminator as TetR without LVA tag. Subsequently the construct was ligated into the pTet-GFP construct, as well. The construct can be found in parts registry as Bba_K1475006.

Characterization/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.

Comparing only the strains expressing either variants of TetR, the results of the FACS illustrates that without induction with doxycycline, GFP is still expressed. Most likely because the promoter is leaky. Despite 100% of the cells being fluorescent in the absence of doxycycline one can see that the fluorescence intensity is markedly reduced in the constructs containing TetR repressor. 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.

Although the FACS results indicates that the pTet inhibited by TetR with LVA tag is the most responsive upon induction by doxycycline, we argue that the effect seen is due to overexpressing of TetR repressor. The hypothesis is based on the poor median fluorescence compared to un-regulated pTet promoter, even at doxycycline concentrations inhibitory of cell growth. pSB1C3 being a high copy plasmid leads to a high number of repressors, thus a higher concentration of doxycycline in needed to induce the expression from pTet. The LVA tag destabilizes TetR thus lovering the number of TetR proteins. This could explain the better response from induction of TetR with LVA. It can be seen from the coomassie stain below that there is less TetR repressor with LVA than without, supporting this hypothesis.

By using a strain, constitutively expressing tetR with pTet on a low copy plasmid UNIPV-Pavia iGEM 2011 shows here: BBa_R0040:Experience that pTet can be induced by aTc. Thus less TetR repressors in comparison to pTet sites increases the response to inducer, futher supporting the hypothesis.

To analyse the amount of TetR with and without LVA tag present in the cell, coomassie 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 degradation.

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

Table 1: Plating of E. coli MG1655 K12 expressing different constructs on plates containing a varying concentration of doxycycline: GFP=BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR no LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR +LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter. The experiment was done in duplicates but the second line of results was omitted from this wiki because the results showed the same, please see the parts registry page for all results: BBa_K1475005 and BBa_K1475006.

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 was also seen from the FACS results. Furthermore, the plating assay proves that the bricks are functional, however slowly responding to induction (continuous induction over 24 hours compared to induction over 1 hour)

To see how the growth of the bacteria expressing GFP controlled by pTet are affected, we 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.