Difference between revisions of "Part:BBa K3739039"

Revision as of 18:15, 21 October 2021

J23100-B0030-LMT-his-CBM-hutH-B0010

LMT is a signal peptide from the lytic murein transglycosylase of V.natriegens, his-tag enable us to purify the linking protein, CBM can bind to cellulose and hutH is a histidine transaminase which converts histidine into urocanic acid
LMT here represents a signal peptide used to secrect the fusion protein outside the cell. 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_xxxx to construct the expression system and to express and to purify the protein.

Biology

LMT
Lytic murein transglycosylase (LMT) is an enzyme which is able to degrade murein, a component of cell wall of bacteria. There are two kind of LMTs existing in E.coli: the membrane-binding one and the soluble one. The gene coding LMT homolog is also incorporated in the genome of Vibrio natriegens. The LMT signal peptide (named LMT in our parts) is from the LMT homolog, which can lead the fused protein secreted out of Vibrio natriegens.

CBM
Cellulose enzymes have two domains, and the one that helps bind to cellulose is called cellulose binding module (CBM), and therefore it helps our fusion protein bind to cellulose-rich cell wall. Here we choose the CBM of CenA from Cellulomonas fimi, which has been successfully expressed in Escherichia coli.

hutH
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-LMT-his-CBM-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_xxxx to construct the expression system and demonstrated of the effect of secretory peptides and CBM-hutH together with BBa_xxxx and BBa_xxxx.

Characterization

References=

1. https://parts.igem.org/Part:BBa_K3185005
2. 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