Part:BBa_K191004:Experience

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Applications of BBa_K191004

To characterize this biobrick (that we also refer to as our read out system n°1), we cultured the cells in a medium without tryptophan: 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 tryptophan 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 tryptophan, 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 tryptophan (simply due to cell growth), compared to a decrease in fluorescence for the samples in which we added tryptophan, since tryptophan would enhance the activity of the tryptophan 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

Sequencing the DNA of this part by the 2011 iGEM Team Freiburg indicates that there is a deletion of TA at position 86&87. This should not have an effect, since the deletion is behind the promotor and in front of the RBS.

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