Ncoi ecori digested ptrchis2b

Example 2

Chromosomal DNA of Lactobacillus reuteri strain F275 was obtained from ATCC (ATCC #23272D-5, ATCC, Manassas, Va.). The gene encoding phosphoketolase (PKL) enzyme was amplified using primers CMP34: 5′-taaggaggaataaacATGGCAGTAGATTACGATTCCAAG-3′ (SEQ ID NO:32) and CMP335: 5′-ttctagaaagcttcgttacttaagacccttccaagtccag-3′ (SEQ ID NO:33), 100 ng DNA as template and the polymerase Herculase II Fusion according to the manufacturer (Agilent, Santa Clara, Calif.). After purification, the 2442 bp fragment was assembled into NcoI/EcoRI-digested pTrcHis2B (Invitrogen, Carlsbad, Calif.) using the GENEART seamless cloning kit (Invitrogen, Carlsbad, Calif.) to form plasmid pCMP1029 (SEQ ID NO:16—FIG. 7).

Example 1

Chromosomal DNA of Bifidobacterium infantis was obtained from ATCC (ATCC #15697D-5, ATCC, Manassas, Va.). The gene encoding phosphoketolase (PKL) enzyme was amplified using primers CMP283: 5′-ctgtatTCATGAcgagtcctgttattggcacc-3′ (SEQ ID NO:30) and CMP284: 5′-ctctatGAATTCTCACTCGTTGTCGCCAGCG-3′ (SEQ ID NO:31), 100 ng DNA as template and the polymerase Herculase II Fusion according to the manufacturer (Agilent, Santa Clara, Calif.). After purification, the 2798 bp fragment was digested with BspHI and EcoRI, and ligated with NcoI/EcoRI-digested pTrcHis2B (Invitrogen, Carlsbad, Calif.) to form plasmid pCMP1090 (SEQ ID NO: 15—FIG. 6).