Team:SDU-Denmark/Tour41
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Revision as of 22:34, 17 October 2014
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.
![](https://static.igem.org/mediawiki/2014/7/72/2014SDUexpressions1.png)
The results of the FACS illustrates that without induction with doxycycline, GFP is still expressed. This is 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 reduces 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.
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. 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 can explain the better signal from induction of TetR+LVA. It can be seen from the coomassie stain below that there is less TetR repressor with LVA than without. Because the cells are in exponential growth phase during FACS the number of ribosomes is high this supports the explanation that expression of TetR is high.
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.
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 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.
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.
![](https://static.igem.org/mediawiki/2014/e/e3/2014SDUexpressions31.png)
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.