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

In the previous section, the expression level of the cold-inducible promoter (Pcold: BBa_K1949000) that triggers the start of the story was examined. What happens if this story is set in warm countries? Therefore, in this section, the expression level of the temperature sensitive promoter (Pheat: BBa_K608351), which is heat-inducible, was examined.

2. Summary of the experiment

The objective of the experiment here is to characterize temperature dependency of Pheat; 28°C and 37°C are non-inducible and inducible conditions for this promoter. We prepared three samples shown below. After temperature up-shift, the RFU of GFP at regular time intervals was measured.

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 was 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 at the indicated time points after temperature up-shift. The error bar represents the standard deviation of three samples which derived from three different colonies, respectively.

The RFU of GFP induced by the promoter was calculated by subtracting the RFU of GFP of negative control at each temperature from the RFU of GFP of Pheat ‐ rbs ‐ gfp. It was confirmed that GFP expression was induced when cultivating at 37°C but not at 28°C. Therefore, it is concluded that Pheat is a heat-inducible promoter.

4. Discussion

GFP was not induced from the Pheat ‐ rbs ‐ gfp plasmid at low temperatures and was induced at high temperatures. This result is consistent with reference [1]. Therefore, this experimental result is considered to be 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