Part:BBa_K3031015

LuxI with FUR box located in the -10 region

This part allows for the repression of the LuxR/LuxI Quroum Sensing system in environments containing iron.

This is the LuxI promoter sequence with a standard Ferric Uptake Regulator sequence (FUR box) inserted at the -10 region. The FUR box allows for the repression of the LuxR/LucI Quroum Sensing system and therefore can control the expression of downstream genes in the presence of ferric iron even at high cell densities. In the classical FUR repression mechanism, iron-bound Fur binds to a Fur box sequence that overlaps with, or is proximal to, promoters of iron responsive genes, thus preventing their transcription. When intracellular iron is depleted, Fe2+ is released from FUR, causing conformational changes in the protein resulting in dissociation from the Fur box.

In the normal LuxI sequence, the promoter works as an autoinducer synthase that produces freely diffusible N-acylhomoserine lactone (AHL) molecules. AHL consists of a homoserine lactone (HSL) ring and an acyl chain that vary in length and degree of saturation. When the AHL reaches a threshold concentration, which increases alongside the cell population density, two molecules of LuxR (under the control of LuxR promoter) bind to two AHL autoinducers and form a complex. These activated compelxes act as a transcription factor of Plux promoter (BBa_R0062) and thus downstream genes are expressed at high cell densities.

In a low iron environment, through the LuxI-Fur box promoter, the LuxI protein (AHL) is produced as normal due to the release of Fe2+ from FUR box. The well characterized and used LuxR/LuxI quorum sensing system works as expected then. Thus in the absence of Fe the downstream genes under control of this part are once gain turned on at high cell densities.

Design of part

Insertion of the FUR box was informed by the report by Chu et al., (2015). This study inserted the FUR box region at different locations on a plasmid containing the LuxR/LuxI QS system. The construct with the insert at the -10 region (termed ironQS2) performed the best in experiments testing different constructs (as seen below) in low iron environments as seen in the data below. We used the sequence from the FUR box part (BBa_K1122069) added to the registry by GEM13_Edinburgh.

Different constructs of ironQS (Chu et al., (2015)). Image showing different designs of ironQS (LuxI with FUR box) Our design followed ironQS2 where the FUR box sequence was inserted into the -10 location of the luxI promoter.

Different constructs of ironQS (Chu et al., (2015)). Image showing different designs of ironQS (LuxI with FUR box) Our design followed ironQS2 where the FUR box sequence was inserted into the -10 location of the luxI promoter.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]