Poster Presentation 3-48

Expression of Green Fluorescent Protein (gfp_uv) by E. coli, for Different Inoculum Densities and Induction Times

Thereza Christina Vessoni Penna, Marina Ishii, Adalberto Pessoa Júnior,

Tatiana Rivas Marante, and Érica Miessi Sanches

Department of Biochemical and Pharmaceutical Technology

School of Pharmaceutical Science

University of São Paulo

Rua Antonio de Macedo Soares, 452, zip code: 04607-000

São Paulo/SP, Brazil

Telephone:  0055-11-38183694; Fax:  0055-11-38156386; E-mail:  tcvpenna@usp.br

Transformed cells of Escherichia coli DH5-a with pGFP, induced by IPTG (isopropyl–b-d-thiogalactopyranoside), express the green fluorescent protein (gfp_uv) simultaneously with growth phases.  To obtain the best expression of gfp_uv during logarithmic cell development, the influence of starting inoculated cultures with a pre-incubation period and adding IPTG at different cell densities was studied.  Isolated 24 h, a brightly fluorescent colony grown on LB/amp (Luria Bertani/ampicillin; 100.0 mg/ml) / IPTG (final concentration, 0.5 mM) agar was transferred to LB/amp broth. The culture was developed (37 ^oC/100 rpm) up to OD_660nm ranging from 0.0054 to 0.0161 (10⁵-10⁶ CFU/mL), when an inoculum of 1.0 mL of the developing culture was transferred to each 25 mL of LB/amp broth.  For a total 8-h growth, (i) flasks of IPTG directly incubated were added at OD_660nm = 0.6–0.8 (10⁸ CFU/mL); (ii) flasks kept at 4 ^oC for 12 h before incubating were added at OD_660nm = 0.0073-0.0436 (10⁵-10⁶ CFU/mL).  The specific rate constant (m, h^-1) ranged as follows: (i) cell growth, from 1.09 to 2.36 h^-1; (ii) released gfp_uv (mg/ml) contents, from 0.74 to 0.89 h^-1, corresponding to 9-13% gfp_uv (mg/mg) extracted from 8-h growth cells; (iii) specific gfp_uv contents from 0.70 to 0.90 h^-1 for mg gfp_uv/ mg total protein, and productivity from 0.40 to 0.50 h^-1 for mg gfp_uv/ mg DCW.  The previous overnight incubation of the culture showed a positive influence in the gfp_uv expression by developing cells induced at the beginning of the logarithmic phase. 

Back to main Symposium page

This page was updated 03/25/02