Part:BBa_K2273114

Beta-lactamase encoding gene penP found in B. subtilis

This gene is a part used in the Beta-Lactam Biosensor project of [http://2017.igem.org/Team:TU_Dresden iGEM Team TU Dresden 2017 (EncaBcillus - It's a trap!)]. It codes for a beta-lactamase found in Bacillus subtilis. Yet there is not much known about the activity and activation of the beta-lactamase PenP in B. subtilis. The highest expression levels seem to be achieved when high salt concentrations occur. [http://www.subtiwiki.uni-goettingen.de/v3/gene/view/713BAB7190E1F86C55103049B29072F00E0DFFB3] PenP belongs to the class of Hydrolases and is able to break down beta-lactam antibiotics. This enzyme also harbours a n-terminal signal peptide sequence and is most likely secreted and therefore localized outside of the cell. [http://www.uniprot.org/uniprot/P39824] To investigate the influence that the presence of the beta-lactamase PenP in B.subtilis has on the sensitivity our biosensor, we analyzed the promoter activity of P_penP under antibiotic stress conditions. We amplified a short and a longer version of this promoter to potentially take into account all regulatory regions upstream of the penP gene.

This part features the RFC25 prefix and suffix to enable translational fusions:

Prefix with	EcoRI, NotI, XbaI, RBS, spacer sequence, Start Codon and NgoMIV	GAATTCGCGGCCGCTTCTAGAAGGAGGTGTCAAAATGGCCGGC
Suffix with	AgeI, Stop Codon, SpeI, NotI and PstI	ACCGGTTAAACTAGTAGCGGCCGCTGCAGA

Sites of restriction enzymes generating compatible overhangs are indicated by sharing one color. (EcoRI and PstI are marked in blue, NotI in green, XbaI and SpeI in red, AgeI and NgoMIV in orange)

Beta-Lactam Biosensor

Worldwide, multidrug-resistant bacteria are on the rise and provoke the intensive search for novel effective compounds. To simplify the search for new antibiotics and to track the antibiotic pollution in water samples, whole-cell biosensors constitute a helpful investigative tool. In this part of EncaBcillus, we developed a functional and independent heterologous [http://2017.igem.org/Team:TU_Dresden/Project/Biosensor Beta-lactam biosensor] in Bacillus subtilis. These specialised cells are capable of sensing a compound of the beta-lactam family and will respond by the production of an easily measurable luminescence signal. We analysed the detection range and sensitivity of the biosensor in response to six different Beta-lactam antibiotics from various subclasses. The evaluated biosensor was then encapsulated into Peptidosomes to proof the concept of our project EncaBcillus. The encapsulation of engineered bacteria allows an simplified handling and increased biosafety, potentially raising the chances for their application in e.g. sewage treatment plants.

The role of the beta-lactamase PenP in resistance against beta-lactam antibiotics

In this project we examined the influence of the presence of PenP on our biosensor`s performance. Therefore we investigated the activity of the P_penP promoter in the presence of different beta-lactam antibiotics and two controls (bacitracin and water) in a plate reader assay. The six beta-lactam antibiotics were: ampicillin, carbenicillin, cefoperazone, cefalexin, cefoxitin and penicillin G.

Table 1: Antibiotic concentrations in [µg µl^-1] (final concentration in the well) used in the plate reader assay.

In pre-tests, we investigated the growth of B. subtilis wild type and the PenP mutant, upon exposure to different concentrations of each tested β-lactam (data not shown). After that, we narrowed these down to two concentrations per antibiotic (see Table 1). Since we did not want to kill our biosensors, we focused on β-lactam concentrations which result in a slight inhibition of growth. These concentrations were used for all following experiments to further characterise our biosensors and the effect of the B. subtilis native β-lactamase (PenP) (see Figure 1). [http://2017.igem.org/Team:TU_Dresden/Experiments Plate reader] experiments were performed in [http://2017.igem.org/Team:TU_Dresden/Experiments Mueller Hinton] (MH) media, induction with the antibiotics was carried out after one hour of incubation at 37˚C in the plate reader. Growth was monitored every five minutes for at least 18h.

We expected a higher growth inhibition with rising antibiotic concentrations. In the penP mutant we expected an increased sensitivity towards the β-lactam antibiotics. Addition of water to the culture should not show any effect on the growth and serves as a control. We included a non beta-lactam (the peptide antibiotic bacitracin) in all our assays to demonstrate the specificity of the biosensor.

Figure 1: Growth curves of W168 (1a-f) and W168 penP::kan^R (2a-f) showing an effect after treatment with the tested antibiotics indicated in the legend above the graphs. Samples were induced after 1 hour (indicated by the black line) with (a) ampicillin, (b) carbenicillin, (c) cefoperazone, (d) cefalexin, (e) cefoxitin as well as with (f) penicillin G. Bacitracin and dH₂O serve as controls. Number 1 and 2 represent the two different concentrations tested of each antibiotic, referring to Table 1. Mean values and standard deviation are depicted from at least three biological replicates. The tested antibiotic concentrations can be taken from Table 1.

Table 2: Antibiotic concentrations in [µg µl^-1] (final concentration in the well) used in all further plate reader experiments.

Figure 1: Luminescence (RLU/OD₆₀₀) output of the P_penP promoter versions, two hours after beta-lactam induction. Strains carrying different PenP promoter versions (long or short / 1 and 2) or with varying genetic backgrounds (either W168 or penP::kan^R / a and b). Mean values and standard deviation are depicted from at least three biological replicates.

The analysis of the induction of P_penP by different β-lactam antibiotics unfolded that this promoter seems to be constitutively active during exponential phase (see Figure 1). As the exact promoter length and potential regulatory regions upstream are still unidentified, two versions (short and long) of the promoter have been examined.
The RLU/OD₆₀₀ values shown in Figure 6 indicate a moderate promoter activity during exponential growth. There is no noticeable difference between the strains with a functional PenP enzyme (a) and the penP mutant (b) (see Figure 1). We could not observe a particular activation by β-lactam antibiotics, which suggests that this enzyme is produced might have other functionalities, too.

Figure 8: Results from the Disk Diffusion Assay with the P_penP(long) reporter strains. Daylight pictures (top row) and luminescence detection using a chemiluminescence dock (with two minutes exposure time). Top right scheme shows the disk layout together with the antibiotics applied in the assay. Please note: only results for the long version of the promoter are shown, as the short version behaves accordingly.

We performed disk diffusion assays with the P_PenP(short and long version) reporter strains and checked if any of the β-lactams would lead to a luminescence signal (Figure 2). Unfortunately, none of the tested substance lead to a notable luminescence. We again could only observe a weak basal promoter activity (as in liquid) with both reporter strains. The measured diameters of the inhibition zones are summarised in Table 2. Taking these results together with the observations in liquid (Figure 6), we can state that the native β-lactamase in B. subtilis dose not respond to any of our tested β-lactams.

Table 2: Measured diameter of the inhibition zones in [mm] from the Disk Diffusion Assay with the P_penP(long) reporter strains.

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