Part:BBa_K1897008

LuxR

This part was made up of promoter BBa_J23119, ribosome binding site (RBS) BBa_B0030, LuxR coding sequence BBa_C0062 and double terminator BBa_B0015.

Usage and Biology

This part allows for the constitutive expression of LuxR protein from the promoter BBa_J23119. The LuxR protein originated from Vibrio fischeri, and is a regulatory protein of the Lux operon. The Lux operon can be transcribed from from the left (operonL) or right (operonR). Transcription of operonR, which consists of LuxICDABE, occurs only in the presence of N-(3-oxo-hexanoyl)-homoserine lactone (AHL), which is an autoinducer produced by LuxI (Shaldel and Baldwin, 1991). This also produces light due to the production of LuxCDABE (Shaldel and Baldwin, 1991). Transcription of operonL leads to the production of LuxR (Shaldel and Baldwin, 1991). In the presence of rising amounts of AHL, LuxR-AHL complexes begin to form, and LuxR undergoes a conformational change that leads to the activation of the Lux operon (Trott, 2000).

This part can be used if you require constitutive production of LuxR to activate production of genes under the control of pLuxR (BBa_R0062).

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 50
  
• 1000
  COMPATIBLE WITH RFC[1000]

Construction of composite LuxR

The full composite LuxR was made from overlap PCR of 3 fragments - namely the "front" fragment, comprising of the biobrick prefix, promoter, RBS, as well as some bases from the LuxR coding sequence; the "middle" fragment, comprising of a little of the RBS, the coding sequence of LuxR, the HA tag as well as a few bases of the terminator sequence; and the "terminator" fragment, comprising of a small part of the LuxR coding sequence, the HA tag, the double terminator and the biobrick suffix.

Verification of composite LuxR

Figure 1: DNA gel electrophoresis photo of overlap PCR to stitch the LuxR fragment with front and terminator fragments. The reaction was successful as seen from the observed band size (approximately 1000 bp) for the complete composite LuxR, which is close to the expected band size of 1034 bp (Box B). Each lane in Box B consists of duplicate overlap PCR reaction for LuxR.

In order to verify that the complete LuxR composite has been made via overlap PCR, DNA gel electrophoresis was performed, and the expected band size was 1034 bp. As seen from Figure 1, the band size of product termed 'Full LuxR' has approximately the correct band size of around 1000 bp.

A western blot was also done to verify that the LuxR protein is produced constitutively, as seen in Figure 2. The expected protein band size is around 30 kDa, which is labelled in Figure 2.

Figure 2: Western blotting using the anti-HA antibody revealed a band between 25 and 37 kDa which is close to the estimated molecular mass of LuxR.