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The results from the Plate Reader Assay can be seen in Figure 1.
 
 
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The analysis of the induction of P<i><sub>penP</sub></i> by different &#946;-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>
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<p>The RLU/OD<sub>600</sub> 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 <b>(a)</b> and the <i>penP</i> mutant <b>(b)</b> (see Figure 1). We could not observe a particular activation by &#946;-lactam antibiotics, which suggests that this enzyme is produced might have other functionalities, too.</p>
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Revision as of 18:57, 31 October 2017


Short version of the Promoter PpenP 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 subtilise. 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 PpenP 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 PpenP 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 PpenP 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::kanR / a and b). Mean values and standard deviation are depicted from at least three biological replicates.

The analysis of the induction of PpenP 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.