Part:BBa_K1949001:Design

Design Notes

A point mutation was applied to the Pcold in order to be easy to clone it. Its 177th base is changed from C to A. A referred article describes as stated below.

“We found occasionally that C–A base substitution at nucleotide number 532 in the 5’-UTR region (nucleotide number is according to GenBank Accession No. M30139) diminished this growth inhibition effect (data not shown), allowing easy cloning of the genes of interest into MCS. We do not know why this mutation diminished growth inhibition effect but the mutation did not affect productivity of proteins (data not shown).”

Nakashima,N and Tamura,T. 2004. Cell-free protein synthesis using cell extract of Pseudomonas fluorescens and CspA promoter. Biochemical and Biophysical Research Communications 319 (2004) 672

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 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.

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.