Team:Tokyo Tech/Promoter Assay/Pcold
2-4-1 Pcold assay
Contents
1. Introduction
“Magic Mirror on the wall, who is the fairest one of all?”
Snow White begins with the scene where Magic Mirror replies that “Snow White is.” If the magic mirror didn’t reply as above, Snow White wouldn’t begin. We tested cold inducible promoter (Pcold) as a starter of Snow White. As a result, we found that cold inducible promoter (Pcold) was induced more at 18°C than at 37°C.
2. Summary of the Experiment
Our purpose of this experiment is to characterize temperature dependence of cold inducible promoter (Pcold) by comparing the fluorescence at 18°C with the fluorescence at 37°C using positive control and negative control. We prepared three samples shown below.
・Pcold-gfp (pSB1C3)
・Positive Cntrol : Pcon-rbs-gfp (pSB1C3)
・Negative Control : empty vector (pSB1C3)
3. Results
RFU of GFP/Turbidity of each sample is measured at 18°C and 37°C. The results are shown below.
Fig. 3-4-1- title
This graph shows RFU of GFP/Turbidity per cells every 1 h for 3 h after temperature induction. The error bar represents the standard deviation of three samples which are derived from three different colonies, respectively.
RFU of GFP/Turbidity of GFP induced by the promoter was calculated by subtracting RFU of GFP/Turbidity of negative control at each temperature from the fluorescence intensities of Pcold-rbs-gfp and Pcon-rbs-gfp.
The proportion of RFU of GFP/Turbidity at 18°C to RFU of GFP/Turbidity at 37°C about each sample was calculated.
Fig. 3-4-1- title
It is found that RFU of GFP/Turbidity of GFP induced by Pcold at 18°C was about eight times as much as RFU of GFP/Turbidity of GFP induced by Pcold at 37°C, and RFU of GFP/Turbidity of GFP induced by Pcon at 18°C was about twice as much as RFU of GFP/Turbidity of GFP induced by Pcon at 37°C. It is concluded that Pcold is a cold inducible promoter compared with Pcon which is induced constantly.
4. Discussion
At high temperature condition, GFP was not induced by Pcold. At low temperature condition, GFP was induced by Pcold. This result is consistent with the reference. Therefore, the result is reasonable.
Snow White project attempts to transform Pcold-rhlI into the Magic Mirror coli and to use the Magic Mirror coli as a starter of the story. The Magic Mirror coli cultivated at 37°C will produce RhlI protein and C4HSL and it will be constructed when Snow White begins at low temperature 18°C. In this way, this cold inducible promoter (Pcold) works as expected when it is transformed into the Magic Mirror coli.
5. Materials and Methods
5-1. Construction
-Strain
All the sample were BL21(DE3) strain
-Plasmids
-Pcold-gfp (pSB1C3)
-Positive Control: Pcon-rbs-gfp (pSB1C3)
-Negative Control: empty vector (pSB1C3)
5-2. Assay Protocol
1. Prepare overnight cultures for each sample in 3 mL LB medium containing chloramphenicol (34 microg / mL) at 37 ºC for 12h.
2. Dilute the overnight cultures in 3 mL fresh LB medium containing chloramphenicol (34 microg/mL) so that the OD600 becomes around 0.05 in triplicate (fresh culture).
3. Incubate the triplicated fresh cultures each at 28ºC so that the OD600 reaches 0.3 to 0.4
4. Incubate the triplicated fresh cultures each in water and ice bath for 2min.
5. Incubate the triplicated fresh cultures each at 18ºC, and 37ºC for each sample for 3 h.
6. Measure the OD and RFU of GFP/Turbidity.
6. Reference
[1] Nakashima N and Tamura T. Cell-free protein synthesis using cell extract of Pseudomonas fluorescence and CspA promoter. Biochemical and Biophysical Research Communications. Biochem Biophys Res Commun. 2004 Jun 25;319(2):671-6.
