For us to show that our Project is working, we primarily are required to show that the RNA Thermometers are showing the necessary RBS property. Our studies and experiments were focused on proving that the RNA Thermometer works and for that we tested the RFP attached RNA Thermometer parts. So let’s take a look…
We started off by transformating with our IbpB – RFP and ROSE – RFP parts to our TOP10 E. coli. We applied the procedure and finally finished off the transformation steps. The next day we took our plates and observed that there were colonies in both IbpB and ROSE however only IbpB was colored red but ROSE was white.
Then we acquired the electrophoresis results, and as it can be seen below, both of the parts were the right base length. The IbpB – RFP part is 917 base pairs in total and ROSE – RFP is 902 base pairs. According to the base length of these parts, the cloning was successful.
Before experimenting in different temperatures, we were required to check if the RNA Thermometers showed RBS functionality. For more accuracy, we also cultivated pure RFP containing bacteria as well. In this measurement, we cultivated our IbpB and ROSE parts plus pure RFP for conformation.
We centrifuged the cultures at 14,000 rpm and acquired the result as you can see below. In this, the IbpB and RFP came out as red however the ROSE part didn’t turn out red. This showed that our IbpB part was correctly showing RBS properties but the ROSE Thermometer didn’t work.
As previously mentioned, RFP is the main factor in calculating the effiency of our RNA Thermometers in various temperatures. For us to find out the amount of RFP produced we prepared several liquid culture specimen.
To examine if the RNA Thermometer works in different temperature conditions, we initially used 2 different temperatures to cultivate our bacteria. We selected the first temperature as a temperature in which the IbpB RNA Thermometer would properly thermosense which was 42 C; we chose the second temperature as one that there would be no RFP translation and that was 25 C.
To ensure that the amount of bacteria that would reproduce would be in equal amounts in both IbpB and RFP, we needed to use the spectrophotometer to calculate the OD. For the OD, we needed to use a value in which the bacteria would reach optimum growth but also not produce significant amount of RFP. We specified this value as 0.3, half the optimum growth value of the bacteria with also RFP production, 0.6.
The cultures that we bring to 0.3 OD value are then seperated for incubation in the 2 different temperatures. The cultures in 42 C were incubated for 24 hours and the cultures in 27 C were incubated for 36 hours.
Through examining the pellets of the cultures, we have reached the consensus that our IbpB Thermometer is temperature sensitive and also shows RBS properties however shows slightly less RBS functionality when compared to the RBS of RFP, B0034.
We transferred the cultures to 96 holed plate. We did a measurement with Western-Biored and the result was the same as our expectations and wishes.
All these steps lead to the following ideas:
The fluorescent microscope was elected to measure the amount of RFP. Bacteria that produced RFP glowed with more intensity and this could be observed with the microscope.
Through taking advantage of the fact that there is extra glowing in RFP containing bacteria, we calculated the difference in red light exposure and prepared them as quantitative data through the use of computer programs.
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