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Monitoring of morphology and rheology during recombinant Escherichia coli cultures

Grant number: 14/11557-9
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): October 01, 2014
Effective date (End): May 24, 2015
Field of knowledge:Engineering - Chemical Engineering
Principal researcher:Teresa Cristina Zangirolami
Grantee:João Victor de Lima Andrade
Home Institution: Centro de Ciências Exatas e de Tecnologia (CCET). Universidade Federal de São Carlos (UFSCAR). São Carlos , SP, Brazil
Associated research grant:08/05207-4 - Pneumococcal conjugate vaccine: capsular polysaccharide - pneumococcal surface protein A, AP.TEM

Abstract

The usage of PspA antigenic protein, from S. pneumoniae surface, as a carrier protein can enhance the immunization capacity of the conjugated vaccine employed in the prevention of diseases caused by this bacterium. The cloning of this protein in E. coli enables its obtainment in large quantities. However, the production of heterologous proteins leads to stress, which can cause cell differentiation and, consequently, change the broth viscosity. This study aims to improve the methodology based on the use of computational resources to characterize the dimension of the cells present in the population as well as to assess the influence of the target protein production upon cell morphology and cultivation broth rheology. The experiments will be carried out in bench scale bioreactor, using defined medium and E. coli BL21(DE3) transformed with pET37b+ plasmid expressing PspA4Pro protein. Lactose and IPTG will be used as inducers, under moderate (27ºC) and accelerate (37ºC) induction conditions. Samples withdrawal throughout the cultures will be employed for routine analysis (concentration of biomass, target protein, glycerol, lactose and organic acids) as well as for assessing the morphology (analysis of slides containing Gram stained cells in optical microscopy followed by digital images acquisition and treatment with ImageJ® and Matlab® software to estimate cell length and diameter) and the rheology (determination of shear rate and stress in concentric cylinder rheometer). The integrated analysis of the results will enable to correlate the induction strategy with the expression level as well as with the changes in the morphology and rheology.

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