Part:BBa_K1897009

LuxI

This part was made up of promoter pLuxR BBa_R0062, ribosome binding site (RBS) BBa_B0030, LuxI coding sequence BBa_C0061 and double terminator BBa_B0015.

Usage and Biology

This part encodes LuxI that is under the control of pLuxR BBa_R0062. It can be used together with BBa_K1897008 to create a quorum sensing system (see BBa_K1897015), where in the presence of high cell density, products under the control of pLuxR will be produced.

The LuxI protein is a 193 amino acid protein that originates from Vibrio fischeri, and produces the autoinducer commonly known as N-3-(oxohexanoyl)homoserine lactone (AHL) (Fuqua et al., 1994). The cell membrane is permeable to AHL, allowing AHL to accumulate in the surrounding environment (Fuqua et al., 1994). In lower cell densities, AHL diffuses out of the cell down a concentration gradient, while in high cell densities, it is able to accumulate within the cell to turn on the transcrption of genes under the control of pLuxR (see: BBa_R0062) or pLuxL (see: BBa_R0063) (Fuqua et al., 1994).

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 869
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 82
  
• 1000
  COMPATIBLE WITH RFC[1000]

Construction of composite LuxI

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

Verification of composite LuxI

Figure 1: DNA gel electrophoresis photo of overlap PCR to stitch the LuxI fragment with front and terminator fragments. The reaction was successful as seen from the observed band size (between 750 and 1000 bp) for the complete composite LuxI, which is close to the expected band size of 936 bp (Box A). Each lane in Box A consists of duplicate overlap PCR reaction for LuxI.

In order to verify that the complete LuxI composite has been made via overlap PCR, DNA gel electrophoresis was performed, and the expected band size was 936 bp. As seen from Figure 1, the band size of product termed 'Full LuxI' has approximately the correct band size.