Part:BBa_K3739012

his-rhlA

Required for rhamnolipid surfactant production.

Biology

The RhlA comes from Pseudomonas aeruginosa, and is capable of competing with the enzymes of the type II fatty acid synthase (FASII) cycle for the β-hydroxyacyl-acyl carrier protein (ACP) pathway intermediates, therefore supplies the acyl moieties for rhamnolipid biosynthesis. RhlA has a greater affinity for 10-carbon substrates, and is the only enzyme required to generate the lipid component of rhamnolipid.¹

Usage

In order to obtain purified RhlA, we add a his-tag (6∗His) at the N-terminal of RhlA. We used BBa_K081005 to construct the expression system and obtained the composite BBa_K3739046, 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 transferring the plasmid into Vibrio natriegens, colony PCR was used to certify the plasmid was correct. The expected result was obtained(1305bp).

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

Reference

1. Zhu, K.; Rock, C. O., RhlA converts beta-hydroxyacyl-acyl carrier protein intermediates in fatty acid synthesis to the beta-hydroxydecanoyl-beta-hydroxydecanoate component of rhamnolipids in Pseudomonas aeruginosa. J Bacteriol 2008, 190 (9), 3147-54.

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 87
  Illegal XhoI site found at 823
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 312
  Illegal BsaI.rc site found at 496