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.

Usage & Biology

Quorum sensing allows bacteria to control gene expression according to their population density. In natural gram negative bacteria, their quorum sensing circuits contain homologues of two Vibrio fischeri, a bioluminescent marine bacterium, regulatory proteins called LuxI (BBa_C0061) and LuxR (BBa_C0062). LuxI is the autoinducer synthase that produce freely diffusible N-acylhomoserine lactone (AHL) molecules, which is consisting of a homoserine lactone (HSL) ring and an acyl chain that vary in length and degree of saturation. When the AHL reach a threshold concentration, which increases alongside the cell population density, two molecules of LuxR bind to two AHL autoinducers and form a complex, the transcription factor of Plux promoter (BBa_R0062). LuxR protein degrade rapidly if binding does not take place. AHL help the LuxR protein to stabilize and fold to activate the transcription of the target gene.

We added this part to the construct below to create a composite part that would repress the downstream expression of genes under the influence of the Lux promoter in low iron environments. This circuit achieved this because with luxI not producing AHL, the auto-inducer for Lux promoter is absent. See experiment for results.

Our new part aimed at repressing the expression of genes under the influence of that Lux promoter even at high cell densities when the cell is is iron starved conditions. This part has applications for designing orally administered vaccine or biomolecule delivery systems in gram negative cells because in many vertebrates the intestines is a low iron environment, allowing our part to be employed in systems to present antigens to the mucosal tissue of the intestines. This provides promise for designing bacterial delivery systems which are capable of protecting their active molecules or antigens through the harsh environment of the animals gastrointestinal tract. Our project designed this system as a vaccine delivery system for koi fish (Cyprinus rubrofuscus). We used this new part in two composite parts: BBa_K3031016 which could test the repression of the LuxR/LuxI QS system in low iron environments by constructing a circuit containing a constitutive promoter expressing LuxR and LuxI under the influence of our new LuxI+FUR promoter. A reporter GFP was constructed downstream of the Lux promoter. Our hope was to only see GFP produced in low iron environments once Fe2+ is released from FUR box and AHL could be produced as normal (see data below under Experiment). We then designed and constructed another composite part which replaced the reported gene GFP with our targeted antigen which was known to stimulate an immune response in Koi fish (a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K3031017">BBa_K3031017).

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.

• Sample A = Blank (E.coliBL21(DE3) cells with no plasmid)
• Sample B = E.coliBL21(DE3) cells containing our ironQS system (BBa_K3031016) and grown in iron rich media PLUS iron chelator 2,2'-Dipyridine
• Sample C = E.coliBL21(DE3) cells containing our ironQS system (BBa_K3031016) and grown in iron rich media only.

Results of ironQS system testing. The graph shows for sample B, when our new luxI+FUR promoter was grown in the presence of 2,2'-Dipyridine, all the Fe2+ is released likely released from FUR and thus N-acylhomoserine lactone (AHL) molecules are produced which bind to LuxR molecules up-regulating the expression of GFP which is under control of the Lux promoter. 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 which allowed for the eventual expression of GFP. In sample C, when E.coli Bl21(DE3) cells were grown in the presence of iron (i.e. no 2,2'-Dipyridine) the ferric iron stays bound to the FUR box sequence and allows for the repression of the LuxR/LucI Quroum Sensing system. Sample A shows the blank control which contained no engineered plasmid and thus the fluorescence from sample A is attributed to normal cell metabolism. The fact that Samples A and C show similar results suggests the repression of the LuxR/luxI system was tightly controlled by ferric iron binding to the fur box and inhibiting the production of AHL. Our measurements show relative fluorescence values of BL21 cell containing ironQS system after cultured in iron-rich and iron-limited media respectively. The Blank strain was set as controls.

Usage and Applications

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]