pFR2

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	pFR2 plasmid DNA was introduced into E. coli B-type strain by electroporation, and transformants were selected at 30°C (permissive temperature) on LB plates supplemented with kanamycin and X-Gal (see Materials and Methods).

Protocol tips
pFR2 plasmid DNA was introduced into E. coli B-type strain by electroporation, and transformants were selected at 30°C (permissive temperature) on LB plates supplemented with kanamycin and X-Gal (see Materials and Methods).

Publication protocol

pFR2 plasmid DNA was introduced into E. coli B-type strain by electroporation, and transformants were selected at 30°C (permissive temperature) on LB plates supplemented with kanamycin and X-Gal (see Materials and Methods). On this medium, cells carrying the plasmid in the episomal form gave blue colonies, and integrants obtained after prolonged cultivation (16–24 h) at non-permissive temperature (44°C) gave white colonies. The genetic stability of the integrants was tested at permissive temperature (30°C) in the presence or absence of kanamycin, as reported in Material and Methods. All clones examined retained the ability to grow in the presence of kanamycin and exhibited a white phenotype on X-gal-containing plate indicating that pFR2 was stably integrated in the lacZ locus. One of these integrants, designated FR12, was chosen for further analysis. The same strategy was used to integrate the catabolic genes in the E. coli K-12 derivatives JM109 and DH5α strain, and the corresponding integrants were designated FR13 and FR14, respectively. Additional PCR amplification of the integration-junction sequences with specific primer sets for right and left junctions confirmed that integration occurred at the correct site (data not shown). Moreover, to exclude the possibility that ech and fcs genes have undergone mutational inactivation after stable integration into the E. coli chromosome, the coding region of each gene was amplified as PCR product, cloned and sequenced. This analysis allowed us to demonstrate that all reported recombinant strains had no alteration in the ferulic catabolic genes and could produce enzymatic activities required for conversion of ferulic acid to vanillin.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Protein Expression Prokaryotic cells - E. coli ferulic acid using pFR2 from Maurizio Ruzzi, Department for Innovation in Biological, Agro-Fo.

Manufacturer protocol

Download the product protocol from Maurizio Ruzzi, Department for Innovation in Biological, Agro-Fo for pFR2 below.

Videos

Check out videos that might be relevant for performing Protein Expression Prokaryotic cells - E. coli ferulic acid using pFR2 from Maurizio Ruzzi, Department for Innovation in Biological, Agro-Fo. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.