Team:Tokyo Tech/The experiments of creating E. coli mutants which secrete protein extracellularly
E. coli mutation secreting proteins extracellularly
Contents
1. Introduction
E. coli has the two membrane: the outer membrane and the inner membrane. Then, E. coli rarely secrets proteins to outside the cell.
2. Summary of the experiment
To put our future work into practice, it is essential to obtain E. coli having the ability of secreting any favorite protein. To obtain such E. coli, we expose UV to MG1655 containing beta-lacetamase, which has signal peptide and be secreted to periplasm. We are working on trying to obtain the mutant having ability of secreting protein to outside the cell.
3. Results
The pictures of the exposed UV colonies below (Fig.5-2-3).
Fig. 5-2-3
4. Material and methods
4-1. Construction
-Strain
All the plasmids were prepared in MG1655 strain.
DH5alpha
-Plasmid
A. Pcon-rfp (pSB6A1)
-Medium
LB medium A:
LB medium containing ampicillin (50 microg/ mL)
4-2. Assay protocol
The following experiments is performed at 37℃ unless otherwise stated.
1) Incubate transformed MG1655 containing plasmid-A with vigorous shaking overnight.
2) Dilute the overnight cultures to 1/100,000 in fresh LB medium A.
3) Streak the fresh cultures on an agar plate containing ampicillin (50 microg/ mL).
4) Expose the plate at 9 or 11 *100 microJ/ cm2 and incubate it overnight.
5) Make a replica of the colonies on the plate with velvet.
6) Pore the soft agar containing ampicillin (50 microg/ mL).
7) Streak DH5alpha without ampicillin resistance on the soft agar and incubate overnight.
8) Observe whether the colonies of DH5alpha are on the soft agar.
5. Reference
(1) F.J.M. Mergulha ̃o et al (2005) Recombinant protein secretion in Escherichia coli. Biotechnology Advances 23 (2005) 177–202
