Difference between revisions of "Part:BBa K1475005"

Revision as of 22:35, 25 October 2014

GFP controlled by TetR and pTet.

This composite part has GFP controlled by a TetR, p(TetR) expression system. The TetR repressor protein binds to tetO operator sequence in the Tet promoter repressing expression. [1] The tet repressor protein is inhibited by Tc (tetracyclin) and it's analogues [http://openwetware.org/wiki/ATc ATc (anhydrotetracycline)] and dox (doxycycline) hereby inducing the expression of GFP.[2]
Please note that this part contains the TetR repressor without the LVA rapid degradation tag, for a similar part containing TetR repressor with LVA tag please see Part:BBa_K1475006. The TetR+LVA bacis part can be found here: Part:BBa_C0040.

Usage and Biology

This part can be used to test the expression from the TetR p(TetR) system.

Sequence and Features

Characterization

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 and is found as BBa_K1475006.

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. [4]

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 fluorescence activated cell sorting (FACS) before and after induction with doxycycline. The strains used are NoTetR=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR:LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter.

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 makedly 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: Part: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.

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.

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.

Plating of E. coli MG1655 K12 expressing different constructs on plates containing a varying concentration of doxycycline: GFP=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR no LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR +LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter.

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.

Growth curve of E. coli K12 MG1655 expressing different constructs in the pSB1C3 vactor: Empty vector=pSB1C3. WT=E. coli K12 MG1655. GFP=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR+LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter.

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.

References

1. C. Krafft, et al.: Interaction of Tet Repressor with Operator DNA and with Tetracycline Studied by Infrared and Raman Spectroscopy. Biophysical Journal, Volume 74, Issue 1, January 1998, Pages 63–71. http://www.sciencedirect.com/science/article/pii/S0006349598777677
2. Tetsystems, 2008: Principles and Components Description. http://www.tetsystems.com/science-technology/principles-components
3. Cormack, B.P., Valdivia, R.H., and S. Falkow. FACS-optimized mutants of green fluorescent protein (GFP). Gene 173: 33-38 (1996). http://www.sciencedirect.com/science/article/pii/0378111995006850
4. 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. http://www.nature.com/mt/journal/v15/n5/full/6300118a.html