Part:BBa_K3739013

his-rhlB

transferase activity, transferring hexosyl groups.

Biology

RhlB

The rhamnosyltransferase (RhlB) comes from Pseudomonas aeruginosa, is capable of catalyzing the reaction between HAA and dTDP-l-rhamnose to form mono-rhamnolipid.1 The hydropathy plot of the RhlB suggested that the protein is membrane-anchored via its N-terminal, crossing the membrane and exposing its domains on both sides of the membrane.2

Usage

In order to obtain purified RhlB, we added a his-tag (6∗His) at the N-terminal of RhlB. We used BBa_K081005 to construct the expression system and obtained the composite BBa_K3739047, which is assembled on the expression vector pET-28a(+) by standard assembly. The constructed plasmid was transformed into Vibrio natriegens, then the positive transformants were selected by kanamycin and confirmed by colony PCR and sequencing.

Characterization

1. Agarose Gel Electrophoresis After receiving the plasmid from Sangon Biotech®, regular PCR was used to certify the correctness of the plasmid. Target bands (1698 bp) can be observed at the position around 1700 bp (Fig. 1).

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

Reference

1. Chong, H.; Li, Q., Microbial production of rhamnolipids: opportunities, challenges and strategies. Microb Cell Fact 2017, 16 (1), 137.

2. Ochsner, U. A.; Fiechter, A.; Reiser, J., Isolation, characterization, and expression in Escherichia coli of the Pseudomonas aeruginosa rhlAB genes encoding a rhamnosyltransferase involved in rhamnolipid biosurfactant synthesis. J Biol Chem 1994, 269 (31), 19787-19795.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1156
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 49
  Illegal NgoMIV site found at 883
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 399