Difference between revisions of "Part:BBa C0040:Experience"
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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 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, | ||
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Revision as of 12:20, 2 November 2014
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UNIQ629983240f6429df-partinfo-00000000-QINU
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SDU-Denmark 2014 |
The effect of doxycycline as an inducer was investigated by the expression of GFP in FACS. 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 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. |
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Aberdeen_Scotland 2009 |
The transformation, miniprep and the gel (undigested and digested with the EcoRI and SpeI) worked as expected. |