Background

Antibiotics represent the most effective treatment against bacterial infections. Since the discovery of penicillin by Alexander Fleming in 1928, many new antibiotics have been constantly developed and were successfully applied to treat life-threatening diseases. This significant advancement in medicine saved millions of lives and still does today. However, fighting microorganisms has never been a completed task, but rather an ongoing race between drug discovery and pathogens developing resistances. Thus, multi-drug resistant bacteria still constitute a major threat for humanity, as infectious diseases represent the second leading cause of death worldwide. [1]

One major reason, for the steady increase of antimicrobial resistances is the “inappropriate use of antimicrobials”. Due to excessive prescription and application in livestock farming, little amounts of antibiotics are nearly found everywhere, even in drinking water. These low-dose and non-lethal concentrations containing habitats, allow bacteria to adjust and develop resistances.

As Beta-lactams make up a large percentage of all antibiotics used, the project preferentially focused on this class of broad-spectrum antibiotics. Carbapenems, penicillin derivatives, cephalosporins and monobactams represent the four main classes of the beta-lactams that sum up to over 100 different active substances. All compounds of this particular group can be easily identified by their common chemical structure: the beta-lactam ring (see Figure1).

To address the increasing development of multi-drug resistant bacteria our iGEM Team aims at developing a novel beta-lactam biosensor in Bacillus subtilis based on the genetics of the bla-operon found in Staphylococcus aureus (for a detailed description consider our Design section below). The genetically engineered Biosensor will help to (I) reliably detect even minimal antibiotic concentrations of compounds from the beta-lactam family in waste and drinking water and (II) unravel producer strains of yet unknown Beta-lactam related antibiotics. After extensive characterization of the detection range and sensitivity, the greater goal is to combine the functional Beta-lactam biosensor with our Peptidosomes. Thereby, we would proof the applicability of EncaBcillus as a completely new cultivation platform. Encapsulation of this whole-cell biosensor, will allow an easier and safer handling of the bacteria and thus making them more appealing for field applications, like for example in sewage treatment plants. As a proof of principle we used six Beta-lactams and two controls (water and bacitracin) to evaluate our Biosensor (table 1).

Design

Genetic engineering

To achieve our goal of encapsulating bacteria into Peptidosomes that can sense antibiotics of the beta-lactam family, we first needed to develop a reliable biosensor strain. In Staphylococcus aureus the bla-operon encodes a one-component system, which is responsible for sensing and mediating resistance against beta-lactam antibiotics. The idea was to transfer the regulatory elements of this operon to Bacillus subtilis and replace the native output – being the beta-lactamase BlaZ – by an easy detectable signal. Thus, making Bacillus subtilis a beta-lactam sensing biosensor. (see Figure 2).

For the creation of our biosensor in B. subtilis, the bla-operon from S. aureus was split into three genetic constructs: (A) The Receptor gene blaR1 under control of a strong constitutive promotor (Pveg), (B) the Repressor gene blaI under control moderate strong constitutive promoter (P_lepA) and (C) the target promoter region of the bla-operon (P_blaZ and P_{blaR1I) in front of the lux-operon (luxABCDE). In addition, an inducible version of the blaR1 construct was made by placing the PxylA promoter upstream of the blaR1 gene (A).}