Team:Guanajuato Mx/Design

iGEM Guanajuato Mx

DESIGN

Biosensor based on Escherichia coli K12 as chassis to detect and kill Pseudomonas aeruginosa

To detect and kill P. aeruginosa we require two modules:

The first module consist of the lasR gene which is expressed constitutively in E. coli K12 (New England Biolabs, Ipswich MA, USA). To control the expression of lasR, the chiA74 (or BtI-BtII) promoters are used (Barboza-Corona et al. 2014). Immediately downstream, the second module, regulated by the lasI promoter (BBa_K649000), controls the expression of the alginate lyase gene and the microcin S gene modified with the signal peptide of the endochitinase ChiA74 from Bacillus thuringiensis (Barboza-Corona et al., 2003). The LasR protein detects the HSL (homoserine lactone), and then binds to the lasI promoter that activates the synthesis of the alginate lyase and microcin S, which are secreted in E. coli as they harbor the signal peptide of ChiA74 (Figure 1). The lasR gene (BBa_C0079), lasI promoter (BBa_K649000) and microcin gene (Bba_K565004) were obtained from iGEM team Biosint_Mexico. The alginate lyase gene was amplified from B. thuringiensis 4Q7 and the ChiA74 signal sequence from the plasmid pEHchiA74 (Castañeda-Ramírez et al., 2013). The ChiA74 signal peptide sequence is functional in E. coli (Castañeda-Ramírez et al., 2013) and allows the secretion of the alginate lyase. All biological parts were assembled using the appropriate restriction enzymes and E. coli was transformed at 2.5 kV in an E. coli pulser (Biorad) using standard transformation protocol.

**Figure 1. Genetic circuit for the detection of P. aeruginosa by quorum sensing, using E. coli K12 as a chassis.** The sensor harbors two modules: The first module (LasR) is used to detect P. aeruginosa, and the second to destroy the biofilm (alginate lyase) and kill (microcin) P. aeruginosa. lasR, encodes the LasR (a sensor protein); HSL, homoserine lactone; chiA74p constitutive promoter in E. coli (or BtI-BtIIp); RBS, Ribosome Binding Site; lasIp promoter that is turned on by LasR-HSL; sp, signal peptide sequence of ChiA74; tt indicate the transcriptional terminator.

Biosensor based on Lactobacillus lactis as chassis to detect and kill P. aeruginosa

We used as chassis a strain of L. lactis UQ2 that is able to produce nisin (an antimicrobial peptide or bacteriocin) This strain was donated for the Dr. Blanca García from the Universidad Autónoma de Querétaro, México. Likewise in E. coli, we designed two modules, the first harbors the genetic elements for detecting P. aeruginosa through the constitutive expression of the sensor protein LasR (Bba_K1365997). The second module harbors the pLasI promoter (BBa_K649001), that controls the expression of an additional copy of nisin (Bba_K1365000) and the alginate lyase. The bacterial biosensor overexpress the antimicrobial peptide and the alginate lyase will be translocated through the cell using the USP45 signal peptide, which is functional in L. lactis (Figure 2).

**Figure 2. Genetic circuit for the detection of P. aeruginosa by quorum sensing, using L. lactis Nis+ as a chassis.** The sensor harbors two modules: the first module (LasR) is used to detect P. aeruginosa, and the second to destroy the biofilm (alginate lyase) and kill (nisin) P. aeruginosa. lasR encodes the LasR (a sensor protein); HSL, homoserine lactone; Cp, constitutive promoter in L. lactis (CP29); RBS, Ribosome Binding Site; lasIp promoter that is turned on by LasR-HSL; sp, signal peptide sequence of USP45; tt indicate the transcriptional terminator.

Description and biopatch design

Our patch consists of a multilayer matrix composed of:
(i) Poly(vinyl alcohol), (ii) petrolatum, (iii) Nutritive Agar + PVA, (iv) Nitrocellulose membrane (0.22 μm) and (v) Autoadherible polymer.

Figure 3. Prototype of prolonged liberation system.