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 the fairest one.” If Magic Mirror did not reply as above, Snow White would not begin.
We analyzed the cold inducible promoter (Pcold: BBa_K1949000) as a starter of Snow White and found that expression from Pcold was induced at 18°C compared to 37°C.
2. Summary of the experiment
The purpose of this experiment is to characterize temperature-dependence of Pcold by comparing the expression level of a reporter gene, gfp, at 18°C with that at 37°C. We prepared three transformants shown below.
A. Pcold ‐ gfp (pSB1C3)
Fig. 3-4-1-2-1 Pcon ‐ gfp (pSB1C3)
B. Positive Cntrol : Pcon ‐ rbs ‐ gfp (pSB1C3)
Fig. 3-4-1-2-2 Pcon ‐ rbs ‐ gfp (pSB1C3)
C. Negative Control : empty vector (pSB1C3)
Fig. 3-4-1-2-3 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. 3-4-1-3-1 Pcold is a cold inducible promoter
Fig. 3-4-1-3-2 Activity of Pcon
These graphs show RFU of GFP / Turbidity per cells at indicated time points after the temperature down-shift. The error bar represents the standard deviation from the triplicated data which are derived from three independent 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 ‐ 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.
Concerning Pcold, it was found that RFU of GFP / Turbidity at 18°C was about eight times as much as that at 37°C. On the other hand, concerning Pcon, RFU of GFP / Turbidity at 18°C was about twice as much as RFU of GFP / Turbidity at 37°C. It is concluded that Pcold is a cold inducible promoter compared with Pcon of which expression is caused constantly.
4. Discussion
Snow White project attempts to insert a Pcold ‐ rhlI plasmid into the Magic Mirror coli and to use the Magic Mirror coli as a starter of the story. The Magic Mirror coli cultured at 37°C hardly produces RhlI protein and C4, which is produced by RhlI. C4 starts to accumulate when Snow White coli is cultured at low temperatures like 18°C. In this way, Pcold works as expected when it is introduced into the Magic Mirror coli.
5. Materials and methods
5-1. Construction
-Strain
All the sample were BL21(DE3) strain
-Plasmids
-Pcold ‐ gfp (pSB1C3)(BBa_K1949001)
-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 37ºC for 12 h.
2. Dilute the overnight cultures in 3 mL fresh LB medium containing chloramphenicol (34 microg/mL) so that the turbidity becomes around 0.05 in triplicate (fresh culture).
3. Incubate the triplicated fresh cultures each at 28ºC so that the turbidity reaches 0.1 to 0.12
4. Incubate the triplicated fresh cultures each in water and ice bath for 2 min.
5. Incubate the triplicated fresh cultures each at 18ºC, and 37ºC for each sample for 3 h.
6. Measure the turbidity 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.
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