Difference between revisions of "Part:BBa K191005:Experience"

Revision as of 15:11, 21 October 2009

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Please enter how you used this part and how it worked out.

Applications of BBa_K191005

The read-out system n°2 is a double-repressor system: there is a Trp promoter in front of the TetR gene, followed by the Tet promoter in front of the RFP gene. This means that it should react both to tryptophane and tetracycline, an allosteric blocker of TetR (for our experiments we used anhydrous tetracycline, ATC).

The experiment this time was done both in LB and M9 media.

Protocol

Cells were cultured overnight in LB medium at 37°C.
Re-inoculated 1 mL of cell culture into 100 mL of fresh medium in Erlenmeyer flasks. Normalized the OD to 0.1 by adding fresh medium. Left to incubate at 37°C for about 2h30.
Normalized the OD to 0.2 by adding fresh medium.
In 2 mL Eppendorf tubes, gently mixed 1.8 mL of cell culture with 0.2 mL of Trp solution, ATC solution or cell culture (negative control). The solutions we used for Trp and ATC had respective concentrations of 8 mg/mL and 500 ng/mL (ATC solution in 50% EtOH).
Loaded 30 ul into each well of the plate for the qPCR machine, with quadruplicates for each condition. The qPCR machine took a measurement of the fluorescence every 3 minutes: this was the data used to obtain the graph you can see further below.

Expected Results

For the negative control (cell culture only), we should see a basal level of RFP expression due to the leakage of the system; this level should remain more or less stable throughout the experiment. On the other hand, both the adding of Trp and ATC should result in an increase in fluorescence. For Trp, its presence in the medium should activate the expression of the Trp repressor, which binds to the Trp promoter, in turn repressing the TetR gene, and so preventing TetR from repressing the RFP gene. Similarly, ATC allosterically blocks TetR, and therefore also prevents it from repressing the RFP gene. In short, Trp and ATC should have a positive effect on RFP expression, thus leading to an increase in fluorescence.

Graphs & Figures

This graph shows the average curve for each condition (tryptophane added, ATC added, or nothing):

RO2 characterization

Analysis & Conclusions

In the graph, you clearly see that after a certain delay, an increase in fluorescence can be noted for the samples where Trp or ATC was added, while the fluorescence of the cells cultured in LB alone remains at a significantly lower level (note the error bars). This confirms that the read-out system reacts as expected, with an increase in fluorescence in response to both ATC and Trp. We can therefore assume that both the Trp promoter and the Tet promoter are functional and that we can use this part in order to characterize the functioning of the LovTap protein.

User Reviews

UNIQ0a50670a9e626561-partinfo-00000000-QINU UNIQ0a50670a9e626561-partinfo-00000001-QINU

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 __NOTOC__
 This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
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 ===Applications of BBa_K191005===
+The read-out system n°2 is a double-repressor system: there is a Trp promoter in front of the TetR gene, followed by the Tet promoter in front of the RFP gene. This means that it should react both to tryptophane and tetracycline, an allosteric blocker of TetR (for our experiments we used anhydrous tetracycline, ATC).
+The experiment this time was done both in LB and M9 media.
+<font size="3">'''Protocol'''</font>
+* Cells were cultured overnight in LB medium at 37°C.
+* Re-inoculated 1 mL of cell culture into 100 mL of fresh medium in Erlenmeyer flasks. Normalized the OD to 0.1 by adding fresh medium. Left to incubate at 37°C for about 2h30.
+* Normalized the OD to 0.2 by adding fresh medium.
+* In 2 mL Eppendorf tubes, gently mixed 1.8 mL of cell culture with 0.2 mL of Trp solution, ATC solution or cell culture (negative control). The solutions we used for Trp and ATC had respective concentrations of 8 mg/mL and 500 ng/mL (ATC solution in 50% EtOH).
+* Loaded 30 ul into each well of the plate for the qPCR machine, with quadruplicates for each condition. The qPCR machine took a measurement of the fluorescence every 3 minutes: this was the data used to obtain the graph you can see further below.
+<font size="3">'''Expected Results'''</font>
+For the negative control (cell culture only), we should see a basal level of RFP expression due to the leakage of the system; this level should remain more or less stable throughout the experiment. On the other hand, both the adding of Trp and ATC should result in an increase in fluorescence. For Trp, its presence in the medium should activate the expression of the Trp repressor, which binds to the Trp promoter, in turn repressing the TetR gene, and so preventing TetR from repressing the RFP gene. Similarly, ATC allosterically blocks TetR, and therefore also prevents it from repressing the RFP gene. In short, Trp and ATC should have a positive effect on RFP expression, thus leading to an increase in fluorescence.
+<font size="3">'''Graphs & Figures'''</font>
+This graph shows the average curve for each condition (tryptophane added, ATC added, or nothing):
+[[Image:iGEM_clone4LB_MeanNew.jpg‎|center|thumb|upright=4|RO2 characterization]]
+<font size="3">'''Analysis & Conclusions'''</font>
+In the graph, you clearly see that after a certain delay, an increase in fluorescence can be noted for the samples where Trp or ATC was added, while the fluorescence of the cells cultured in LB alone remains at a significantly lower level (note the error bars). This confirms that the read-out system reacts as expected, with an increase in fluorescence in response to both ATC and Trp. We can therefore assume that both the Trp promoter and the Tet promoter are functional and that we can use this part in order to characterize the functioning of the LovTap protein.
 ===User Reviews===