Part:BBa_K1897015

LuxR + LuxI

This part is made by 3A ligation (see: https://parts.igem.org/Help:Assembly/3A_Assembly) of LuxR BBa_K1897008 and LuxI BBa_K1897009. Both LuxR and LuxI are derived from Vibrio fischeri.

Usage and Biology

This quorum sensing system was derived from Vibrio fischeri, and involves the regulatory LuxR and the autoinducer producing LuxI. Originally in Vibrio fischeri, 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, which negatively regulates it's own transcription (Shaldel and Baldwin, 1991).

As the cell membrane is permeable to AHL, it diffuses out of the cell down a concentration gradient when there is low cell density (Fuqua et al., 1994). 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).This is due to rising amounts of AHL, which results in LuxR-AHL complexes begin to form, and LuxR undergoes a conformational change (likely to form a dimer) that leads to the activation of the Lux operon (Choi and Greenberg, 1992; Trott, 2000).

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
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1852
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 50
  Illegal AgeI site found at 1065
  
• 1000
  COMPATIBLE WITH RFC[1000]

Construction of LuxR+LuxI

LuxR+LuxI was made via 3A ligation of LuxR BBa_K1897008 and LuxI BBa_K1897009. This was done using the restriction enzyme sites SpeI on the biobrick suffix of LuxR (BBa_K1897008) and XbaI on the biobrick prefix of LuxI (BBa_K1897009). After cutting those two restriction enzyme site, ligation was performed using T4 ligase and a scar site is formed, joining the two fragments together. At the same time, the EcoRI site on the biobrick prefix of LuxR (BBa_K1897008) and the PstI site on the biobrick suffix of LuxI (BBa_K1897009) was also cut, and joined to a pSB1K3 backbone that was also cut with EcoRI and PstI. This resulted in an end product of a plasmid with LuxR and LuxI joined together.

Verification of LuxR+LuxI

Figure 1: Gel electrophoresis photo to check that the 3A ligation was successful for luxR+luxI. A restriction enzyme (RE) digestion was performed using EcoRI and SpeI on the individual plasmids. A single digest control was done to ensure that the digestion was successful. The expected band size for luxR+luxI was around 1900 bp, which is seen in Box A (a band of approximately 2000 bp) on the gel. Each lane in Box A is a duplicate digest reaction.

A restriction digest (single cut as positive control and double cut) was done on the plasmid obtained to check for the insert size. The expected insert size was around 1900 bp, which is shown in Box A of Figure 1. The band above Box A is the pSB1K3 backbone. As the band size appears to be close to 2000 bp, this indicates that the 3A ligation was successful.

Figure 2: Western blotting using the anti-HA antibody revealed bands at approximately 25,30, 60 and 90 kDa which is deduced to be LuxI, LuxR, LuxR dimer and LuxR trimers.

A western blot was also done (Figure 2) to check that LuxR and LuxI are produced. Since this is done in the presence of AHL (produced by LuxI), this likely led to the formation of LuxR dimers (Choi and Greenberg, 1992) and even trimers.