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 tested different constructs with the FUR box region inserted 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.

Performance of each of the different constructs of ironQS (Chu et al., (2015)). IronQS2 with the FUR box site inserted into the -10 region performed the best at regulating downstream genes in iron starved environments.

Experiment

To test the effectiveness of our new part luxI promoter with FUR - we needed to expose cells containing transformed plasmid into both iron rich and iron starved environments. Single colonies were inoculated in 50 ml LB broth containing Ampicillin in a 1000:1 ratio and 40 μM FeSO4 in Falcon tubes and cultured at 37 C until OD600 = 0.5. 10ml culture was added to each of three 15ml tubes. Sample A contains blank cell (without plasmid) culture. Sample B contains culture (with plasmid) with 200 μM DP (2,2'-Dipyridine). The function of the 2,2'-Dipyridine is to remove iron in the cellular environment and thus mimic the low iron environment of the gut. Sample C contains only the culture (with plasmid) without any 2,2'-Dipyridine.

After induction with DP for 4 hours, 1 ml of each cell culture broth was transferred to two 1.5 ml sterile centrifuge tubes and centrifuged at 4000rpm for 4 minutes. After removing the supernatant, we wash the cell with PBS buffer. Then, 100 μM culture was added into 96 well white polystyrene microplate and black polystyrene microplate, each with three samples. We measured the OD600 and Fluorescence (Excitation: 485nm/ Emission: 528nm) by using plate reader. The data was recorded. After that, we calculate the average OD600 and Fluorescence for each sample. For each of samples, we divided the relative fluorescence value (RFV) by the average OD600. This quantitative test was used to determine Fur and luxI/luxR-controlled protein expression under iron deprivation in E. coli.