Part:BBa_K3031016

Iron QS system + GFP reporter

This system is designed to express downstream genes of interest only in low iron environments. The LuxI/LuxR QS system ensures only expression of proteins once a high cell density is reached. We have used the modified LuxI promoter designed by this team to regulate expression of downstream genes using a Ferric Uptake Regulator repressor. The QS system is now inhibited as long as ferric iron is available to the cell as LuxI protein is not produced, resulting in no AHL + LuxR complex forming. Therefore the Lux promoter (pLux) is not activated.

Activation in the system is achieved in low iron environments such as the guts of fish. This makes the system ideal for an oral vaccine delivery system where antigen production could be controlled until the cell reaches the gut of the fish after ingestion.

This system was used to test the low iron environment. We performed an experiment to test the working of our ironQS system and measured its ability to first be controlled by the amount of iron in vitro by measuring fluorescence of GFP under the control of our new luxI + FUR box promoter <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K3031015">BBa_K3031015</a>.

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.

The addition of the Ferric Uptake Regulator sequence into the luxI promoter (our new basic part BBa_K3031015) means that this system can be used to repress downstream expression of genes under the control of the lux promoter even at high cell densities. Our system has applications for designing orally administered vaccine or biomolecule delivery systems in gram negative cells. The reason it is ideal for an oral vaccine delivery system is that antigen production could be controlled until the cell reaches the gut of the organism after ingestion. 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 first tested this part (with GFP) to assess its ability to suppress the LuxR/LuxI QS expression in low iron environments (see data below). We then designed and constructed another composite part which replaced the reported gene GFP with our targeted antigen (part BBa_K3031017).

Overall aim of these parts was to develop a system where a targeted antigen can be controlled by QS and iron starvation which mimics the in vivo conditions of the koi's second gut. This system was designed with the intention to be used in gram negative bacteria such as E.coli or Edwardsiella tarda which has been used successfully as bacterial vaccine delivery strains in previous studies (see references). However we also believe this system can be applied into mice models and other vertebrates where orally administered vaccines are showing promise

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.