Project motivation and description

Multi-resistant bacteria cause millions of deaths each year. While the search for new antibiotics to fight strains who are resistant to broadly used antibiotics continues, we as a team wanted to focus on one cause for multi resistant bacteria. The agricultural industry pollutes the environment with antibiotics on a large scale. To take correct measures to counteract the pollution, detecting antibiotics in samples with an easy-to-use sensor, is one of the first steps to lessen the burden intensive industries put on our planet each year.

The iGEM Bochum Team strives to find a biosensor for beta-lactam antibiotics that are broadly used in the meat industry. The basic idea is to detect the presence of beta-lactam antibiotics in small samples using a whole-cell biosensor, focusing on the analysis of wastewater.

The presence of beta-lactam antibiotics is detected by the sensor through uptake of the antibiotics by the cells, leading to induced expression of the gene for the fusion protein consisting of a beta-lactamase and super fold GFP (sfGFP). This will lead to the degradation of the beta lactam ring by the beta-lactamase and a green, fluorescent feedback. Subsequently, the degradation product induces the expression of another fluorescence protein gene, the mCherry, which provides additional feedback on the degradation of the beta lactam antibiotics in the sample.

To achieve this task, we plan on performing three general steps. We decided to use ,,Bacillus licheniformis,, as our host organism. ,,B. licheniformis,, is a gram-positive bacterium, labelled as a GRAS organism, that possesses a chromosomally localized beta-lactamase gene. This makes the bacterium attractive for the use as a biosensor. First, we want to construct a plasmid that allows us to insert the sfGFP gene chromosomally downstream of the gene for the beta-lactamase by homologous recombination. This is done by cloning 1.5 kb upstream and downstream from the site where the sfGFP is to be inserted chromosomally and the sfGFP gene into the vector pDM4. The cloning is performed via the Gibson-Assembly. The second step involves transformation of the plasmid into ,,B. licheniformis,, by electroporation and the two-step allelic exchange. After transformation of the suicide vector in the host cell, homologous recombination occurs between the allelic exchange vector pDM4 and the recipient chromosome. The success of the first crossover is determined by antibiotic resistance, followed by sucrose counter-selection for the second crossover. Finally, the gene encoding mCherry is placed under the control of a promoter sensitive to proline derivatives, cloned into a plasmid, and transformed into ,,B. licheniformis,,. This leads to the expression of mCherry when beta-lactam antibiotics are degraded, since the degradation product of beta-lactam antibiotics forms a proline analogue.

In addition to our goal of testing for beta-lactam antibiotics, we also have the idea of widening the range of our biosensor by creating different strains that are sensitive to different antibiotics. These strains can be immobilized next to each other on a plate to have one biosensor that detects a vast range of antibiotics.