Difference between revisions of "Part:BBa K3739107"
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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-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. | 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-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. | ||
| − | Here, we used <partinfo>BBa_K3739009<partinfo> to construct the expression system and demonstrated of the effect of secretory peptides and CBM-hutH together with <partinfo>BBa_K3739104</partinfo> and <partinfo>BBa_K3739031</partinfo>. | + | Here, we used <partinfo>BBa_K3739009</partinfo> to construct the expression system and demonstrated of the effect of secretory peptides and CBM-hutH together with <partinfo>BBa_K3739104</partinfo> and <partinfo>BBa_K3739031</partinfo>. |
Revision as of 21:57, 21 October 2021
K525998(T7-RBS)-LMT-his-hutH-B0010
LMT here represents a signal peptide used to secrect the fusion protein outside the cell. The enzyme catalyzes the reaction of converting histidine to form toxic urocanic acid and his-tag is added to purify the protein. We use BBa_K3739107 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.
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-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.
Here, we used BBa_K3739009 to construct the expression system and demonstrated of the effect of secretory peptides and CBM-hutH together with BBa_K3739104 and BBa_K3739031.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 257
Illegal NgoMIV site found at 693
Illegal NgoMIV site found at 1428
Illegal NgoMIV site found at 1664
Illegal AgeI site found at 330
Illegal AgeI site found at 1157
Illegal AgeI site found at 1653 - 1000COMPATIBLE WITH RFC[1000]