Part:BBa_K2273116

Long version of the Promoter P_penP found in B. subtilis

This Promoter 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 controls the gene expression of the penP gene that 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. The enzyme PenP harbours a signal peptide sequence and is most likely secreted and 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 RFC10 prefix and suffix:

Prefix with	EcoRI, NotI, XbaI	GAATTCGCGGCCGCTTCTAGA
Suffix with	SpeI, NotI and PstI	ACTAGTAGCGGCCGCTGCAGA

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)

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. The tested antibiotic concentrations can be taken from Table 1.

Table 1: 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.</p>

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.

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

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.

References:

1 C. Lee Ventola, MS (2015) The antibiotic resistance crisis: part 2: management strategies and new agents. Pharmacy and Therapeutics 40(5), 344–352 2 www.aerzteblatt.de, visited 08/23/17 (5:34pm) [http://www.who.int/mediacentre/factsheets/fs194/en/ 3] www.who.int, visited 09/04/17 (3:21pm) 4 https://en.wikipedia.org/wiki/Β-lactam_antibiotic, visited 10/27/17 (4:42pm) 5 Leticia I. Llarrull, Mary Prorok, and Shahriar Mobashery (2010) Binding of the Gene Repressor BlaI to the bla Operon in Methicillin-Resistant Staphylococcus aureus. Biochemistry 49(37), 7975–7977 Radeck, J., Kraft, K., Bartels, J., Cikovic, T., Dürr, F., Emenegger, J., Kelterborn, S., Sauer, C., Fritz, G., Gebhard, S., and Mascher, T. (2013) 6 The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis. J Biol Eng 7(29),7 Toth, M., Antunes, N.T., Stewart, N.K., Frase, H., Bhattacharya, M., Smith, C. and Vakulenko, S. (2016) Class D β-lactamases do exist in Gram-positive bacteria. Nature Chemical Biology 12(1),9-14