Difference between revisions of "Part:BBa K3739031"

Revision as of 20:57, 21 October 2021

J23100-B0030-his-hutH-B0010

The fusion protein CBM-hutH works on the surface of single cell Phaeocystis. globosa and the enzyme catalyzes the reaction of converting histidine to form toxic urocanic acid. His-tag is added to purify the protein. We use BBa_K3739031 to construct the expression system and to express and to purify the protein.

Biology

The HutH comes from Pseudomonas putida. Under natural conditions, many microorganisms can use the histidine ammonia-lyase (HutH) to change L-histidine into urocanic acid. HutH catalyzes the first step in the degradation of histidine, and the product urocanic acid is further metabolized to glutamate. This enzyme could be found in the liver of vertebrates and in bacteria such as Escherichia coli, Salmonella and Pseudomonas. It is specific for L-histidine and can be inhibited by D-histidine or imidazole. The active center of the enzyme is thought to be dehydroalanine.

Usage

In order to easily purify HutH, we added a his-tag (6*His) at the C-terminal. We ligased the strong promoter (BBa_J23100) and the parts (RBS-his-hutH-Terminator) on the expression vector pET-28a (+) by standard assembly. Then the ligation mixture was transformed into E. coli DH5α, and the correct recombinant one was confirmed by kanamycin, colony PCR and sequencing. We used BBa_ K3739031 to construct the expression system and demonstrated of the effect of secretory peptides and CBM-hutH together with BBa_K3739104 and BBa_K3739107.

Characterization

1. Agarose Gel Electrophoresis When we were building this circuit, colony PCR was used to certify the plasmid was correct. We got the target (1877bp).

Fig.1 The result of colony PCR. Plasmid pET-28a (+).

References

1. Meng, Q. et al. Characterization and enhanced extracellular overexpression of a new salt-activated alginate lyase. Journal of the science of food and agriculture 101, 5154-5162, doi:10.1002/jsfa.11161 (2021).

Sequence and Features