Part:BBa_K1949000:Experience

Materials and Methods

Construction

-Strain

All the sample were BL21(DE3) strain

-Plasmids

-Pcold-gfp (pSB1C3)

-Positive Control: Pcon-rbs-gfp (pSB1C3)

-Negative Control: empty vector (pSB1C3)

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 the fluorescence intensity.

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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