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 start of 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 heat-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 RFU of GFP at regular time intervals.

A. Pheat ‐ rbs ‐ gfp (pSB1C3)

Fig. 3-4-2-2-1 Pheat ‐ rbs ‐ gfp (pSB1C3)

B. Positive Cntrol : Pcon ‐ rbs ‐ gfp (pSB1C3)

Fig. 3-4-2-2-2 pSB1C3

C. Negative Control : empty vector (pSB1C3)

3. Results

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

Fig. 3-4-2-3-1 Pheat is a heat inducible promoter

This graph shows RFU of GFP / Turbidity 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 RFU of GFP induced by the promoter is calculated by subtracted the RFU of GFP of negative control at each temperature from the RFU of GFP of Pheat ‐ 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 heat-inducible promoter.

4. Discussion

GFP is not induced of Pheat ‐ rbs ‐ gfp under low temperature and is induced under high temprature condition. This result is consistent with reference [1]. Therefore this experiment result 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 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 28℃ so that the turbidity reaches 0.1 to 0.12.

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

5. Measure the turbidity and the RFU of GFP.

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