Part:BBa_K1475003:Experience

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•••• SDU-Denmark

The effect of doxycycline as an inducer was investigated by the expression of GFP in FACS. Results of the fluorescence activated cell sorting (FACS) before and after induction with doxycycline. The strain used is E. coli K12 MG1655 with different constructs NoTetR=BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR=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 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 stained SDS-page below that there is less TetR repressor with LVA than without, supporting this hypothesis. The coomassie stained SDS-page 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. 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 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) 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 results found from the plates were quantified by analyzing samples on FACS. Duplicates were made for each sample. Results of the FACS analysis on E. coli K12 MG1655 constructs (No TetR, TetR+LVA and TetR) expressing GFP at various concentrations of doxycycline. The results from the FACS underline what was seen on the plates. It is observed that the induction with increasing doxycycline concentrations does indeed cause an increased expression of GFP. The results for the constitutive GFP expression indicate, as expected, that higher concentrations of doxycycline cause decreased GFP expression, presumably due to cell stress. The same effect might be expected for the two types of TetR constructs. The increased GFP expression is therefor most likely greater than would be seen, if the stress from the doxycycline was absent. The FACS results also supports what was observed from the earlier experiments, that the repressor is leaky, 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. Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.

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