Final Activity Report Summary - BIFIDOGT (Towards gene therapy using Bifidobacterium as gene delivery system)
The main objective of this project for its 1st year was the development of an efficient expression vector for Bifidobacterium, allowing the use of these bacteria in gene therapy as non-viral gene delivery system of prophylactic and/or therapeutic substances in the human intestine or into tumours, eliminating the bottleneck that the lack of this new innovative molecular tool is producing to underpin applications to human health using these probiotic GRAS status bacteria included in the lactic acid bacteria group.
Modular Expression Vector Design:
This task was achieved completely, expression vector of this project have been designed to permit an easy molecular manipulation and cloning, for the study of the expression elements that can affect the expression in Bifidobacterium of proteins of prophylactic and/or therapeutic interest. This expression vector have been designed also to be used as base to develop a set of food grade expression systems and inducible expression vectors using a chromosome-plasmid balanced lethal system as biological containment system in future projects.
Construction of the base expression vector:
This task has been achieved completely with non-remarkable problems. Module 1, Module 2, Module3 (for both selective markers) and Module 4 have been constructed using a PCR approach. Also, Supermodule A (for both selective markers) and Supermodule B have been constructed using a PCR approach.
Identification and experimental corroboration of signal peptides:
This task has been achieved only partially at the moment, using online bioinformatics tools as SignalP V2.0 and the BDGP: Neural Network Promoter Prediction - we have been able to identify a putative signal peptide, and a putative constitutive promoter respectively, searching in the genome of a strain of B. breve. The selection of the putative signal peptides identified have been made in base of the most favourable maximum cleavage score, maximum signal score, maximum combined score and mean signal score, non-alternative processing sites in the cleavage region and have been selected from a exported protein family. The putative promoter and the putative signal peptide have been cloned in frame to construct Module 3 that have been used for construction Supermodule B. Final functional experimental corroboration for signal promoter has been delayed until the construction of the expression vector.
A synthetic gene codifying for Mus musculus cytokine interleukine-10 (mIL-10) with codon usage optimisated for Bifidobacterium has been designed.
A synthetic gene codifying for protein endostatin with codon usage optimisated for Bifidobacterium have been designed.
Modular Expression Vector Design:
This task was achieved completely, expression vector of this project have been designed to permit an easy molecular manipulation and cloning, for the study of the expression elements that can affect the expression in Bifidobacterium of proteins of prophylactic and/or therapeutic interest. This expression vector have been designed also to be used as base to develop a set of food grade expression systems and inducible expression vectors using a chromosome-plasmid balanced lethal system as biological containment system in future projects.
Construction of the base expression vector:
This task has been achieved completely with non-remarkable problems. Module 1, Module 2, Module3 (for both selective markers) and Module 4 have been constructed using a PCR approach. Also, Supermodule A (for both selective markers) and Supermodule B have been constructed using a PCR approach.
Identification and experimental corroboration of signal peptides:
This task has been achieved only partially at the moment, using online bioinformatics tools as SignalP V2.0 and the BDGP: Neural Network Promoter Prediction - we have been able to identify a putative signal peptide, and a putative constitutive promoter respectively, searching in the genome of a strain of B. breve. The selection of the putative signal peptides identified have been made in base of the most favourable maximum cleavage score, maximum signal score, maximum combined score and mean signal score, non-alternative processing sites in the cleavage region and have been selected from a exported protein family. The putative promoter and the putative signal peptide have been cloned in frame to construct Module 3 that have been used for construction Supermodule B. Final functional experimental corroboration for signal promoter has been delayed until the construction of the expression vector.
A synthetic gene codifying for Mus musculus cytokine interleukine-10 (mIL-10) with codon usage optimisated for Bifidobacterium has been designed.
A synthetic gene codifying for protein endostatin with codon usage optimisated for Bifidobacterium have been designed.