Team:Tokyo Tech/Promoter Assay/Pheat

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2-4-2 Pheat assay

Contents

1. Introduction

2. Summary of the Experiment

3. Results

4. Discussion

5. Materials and Methods

   5-1. Construction

   5-2. Assay Protocol

6. Reference

1. Introduction

We tested that the expression level of cold-inducible promoter (Pcold: BBa_K1949000) that controls the story. What happens if this story is set in a warm country? We tested that the expression level of temperature sensitive promoter (Pheat: BBa_K608351), which is high temperature inducible promoter.

2. Summary of the Experiment

Our objective is to characterize temperature dependency of Pheat at 28°C and 37°C with positive control and negative control. We prepared three samples shown below. After temperature induction, we measured the fluorescence intensity at regular time intervals.
・Pheat - rbs - gfp (pSB1C3)
・Positive Cntrol : Pcon - rbs - gfp (pSB1C3)
・Negative Control : empty vector (pSB1C3)

3. Results

The fluorescence intensity of each sample is measured at 28°C and 37°C. The results are shown below.

Fig. 3-5-1- title

This graph shows the fluorescence intensity per cells every 1 h for 3 h that after temperature induction. The error bar represents the standard deviation of two samples which derived from two different colonies, respectively.

The fluorescence intensity of GFP induced by the promoter is calculated by subtracted the fluorescence intensity of negative control at each temperature from the fluorescence intensities of Pheat-rbs-gfp and Pcon-rbs-gfp. It is confirmed that GFP expression can be expressed when cultivating at 37°C but not at 28°C. Therefore, it is concluded that Pheat is a high temperature inducible promoter.

4. Discussion

GFP is not induced of Pheat-rbs -gfp under low temperature and is induced under high temprature. This result is consistent with reference [1]. Therefore this experiment results is considered reasonable.

5. Materials and Methods

5-1. Construction

-Strain
All the sample were BL21(DE3) strain

-Plasmids
-Pheat - rbs - 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 28°C 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 28℃ so that the OD600 reaches 0.3 to 0.4.

4. Incubate the triplicated fresh cultures for each sample at 28ºC and 37ºC for 3 h.

5. Measure the OD and the fluorescence intensity.

6. Reference

[1] M Mieschendahl, B Müller-Hill. F'-coded, temperature-sensitive lambda cI857 repressor gene for easy construction and regulation of lambda promoter-dependent expression systems. J Bacteriol. 1985 Dec;164(3):1366-9.
Freiburg 2011