Difference between revisions of "Part:BBa K191004:Experience"

Revision as of 15:10, 21 October 2009

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

Applications of BBa_K191004

Our first read-out system consists of the Trp promoter followed by the RFP gene. In E. coli, the Trp promoter is situated in front of the Trp operon, which contains the genes necessary for the tryptophane biosynthetic pathway. In brief, genes placed after the Trp promoter should be repressed in the presence of tryptophane.

To characterize this biobrick, we cultured the cells in a medium without tryptophane: M9 medium to which we added all other amino acids and thiamine. You can find the protocol for the M9 medium [http://2009.igem.org/wiki/index.php?title=Team:EPF-Lausanne/Protocols/M9medium here]. The purpose of the experiment was to see the difference of RFP expression depending on whether we added tryptophane to the medium or not.

Protocol

Cells containing the read-out system were cultured overnight in M9 medium.
On the next day, re-inoculated 0.75 mL of cell culture into 25 mL of fresh medium in an Erlenmeyer flask. Incubated for 2 hours at 37°C.
Normalized the OD of all flasks to 0.06 by adding the appropriate amount of fresh medium.
Prepared the plate for the measurements with the qPCR machine, 50 ul per well. For the tryptophane, we used a 4.25 g/L solution. For ATC, it was a 500 ng/mL solution in 50% EtOH, and for IPTG, we always added it so as to have a final concentration of 1mM. The conditions we tested were 1/2 Trp, 1 Trp and 3/2 Trp, 1/2 corresponding to 1 ul.
The plate was then loaded into the qPCR machine, which took measurements of red fluorescence every 3 minutes, giving the graphs you can see below.

Please note that there was a time lag between when we started loading the wells on the plate and when the measurements began, so the induction might already have started.

Expected Results

In this experiment, we would expect to see a slight increase in fluorescence for the cells without tryptophane (simply due to cell growth), compared to a decrease in fluorescence for the samples in which we added tryptophane, since tryptophane would enhance the activity of the tryptophane repressor, which in turn would repress the RFP gene via the Trp promoter. Also to be noted, the RFP decay shouldn't be too abrupt, considering that the RFP protein used doesn't contain a degradation tag.

Graphs & Figures

RO1 final plot

Analysis & Conclusions

We can see that there is a decrease in RFP expression when Trp is added to the medium versus a stable expression for the -Trp condition, which corresponds to what we expected. From this we can therefore conclude that our first read-out system is functional, confirming that the sequence for the Trp promoter seems to be correct.

User Reviews

UNIQ53893f07735c5cdb-partinfo-00000000-QINU UNIQ53893f07735c5cdb-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_K191004===
+Our first read-out system consists of the Trp promoter followed by the RFP gene. In E. coli, the Trp promoter is situated in front of the Trp operon, which contains the genes necessary for the tryptophane biosynthetic pathway. In brief, genes placed after the Trp promoter should be repressed in the presence of tryptophane.
+To characterize this biobrick, we cultured the cells in a medium without tryptophane: M9 medium to which we added all other amino acids and thiamine. You can find the protocol for the M9 medium [http://2009.igem.org/wiki/index.php?title=Team:EPF-Lausanne/Protocols/M9medium here]. The purpose of the experiment was to see the difference of RFP expression depending on whether we added tryptophane to the medium or not.
+<font size="3">'''Protocol'''</font>
+* Cells containing the read-out system were cultured overnight in M9 medium.
+* On the next day, re-inoculated 0.75 mL of cell culture into 25 mL of fresh medium in an Erlenmeyer flask. Incubated for 2 hours at 37°C.
+* Normalized the OD of all flasks to 0.06 by adding the appropriate amount of fresh medium.
+* Prepared the plate for the measurements with the qPCR machine, 50 ul per well. For the tryptophane, we used a 4.25 g/L solution. For ATC, it was a 500 ng/mL solution in 50% EtOH, and for IPTG, we always added it so as to have a final concentration of 1mM. The conditions we tested were 1/2 Trp, 1 Trp and 3/2 Trp, 1/2 corresponding to 1 ul.
+* The plate was then loaded into the qPCR machine, which took measurements of red fluorescence every 3 minutes, giving the graphs you can see below.
+Please note that there was a time lag between when we started loading the wells on the plate and when the measurements began, so the induction might already have started.
+<font size="3">'''Expected Results'''</font>
+In this experiment, we would expect to see a slight increase in fluorescence for the cells without tryptophane (simply due to cell growth), compared to a decrease in fluorescence for the samples in which we added tryptophane, since tryptophane would enhance the activity of the tryptophane repressor, which in turn would repress the RFP gene via the Trp promoter. Also to be noted, the RFP decay shouldn't be too abrupt, considering that the RFP protein used doesn't contain a degradation tag.
+<font size="3">'''Graphs & Figures'''</font>
+[[Image:RO1final.jpg‎|center|thumb|upright=4|RO1 final plot]]
+<font size="3">'''Analysis & Conclusions'''</font>
+We can see that there is a decrease in RFP expression when Trp is added to the medium versus a stable expression for the -Trp condition, which corresponds to what we expected. From this we can therefore conclude that our first read-out system is functional, confirming that the sequence for the Trp promoter seems to be correct.
 ===User Reviews===