iGEM-Engineering

Project design/Engineering In view of the increasing risk of multi-resistant bacteria due to the improper dispensing of antibiotics, we have made it our business to detect antibiotics in wastewater and break them down. We are initially focusing on beta-lactam antibiotics, as these are among the most used antibiotics worldwide. Our sensor must fulfill the following requirements: First, it has to be able to recognize beta-lactam antibiotics, whereupon signal transduction takes place and expression of the reporter gene begins. Second, the reporter must be visually recognizable. Third, the antibiotic must be degraded.

The workflow can be divided into three steps: First, the construction of the plasmid. Second, the integration of the sfGFP into the genome by homologous recombination. Third, the testing of the biosensor.

1. Construction of the plasmid

For homologous recombination, the vector pDM4 is designed for allelic exchange. The vector contains the gene for sfGFP and the 1.5 kbp flanking upstream and downstream DNA regions homologous to the recipient chromosome.

The plasmid is constructed using Gibson Assembly. The primers for amplification of the gene fragments and vector are designed with overlapping overhangs. After amplification, the DNA fragments and vector are assembled in-vitro.

2. Integration of sfGFP into the genome The plasmid is transformed into E. coli S17 using the heat shock method. Uptake of the plasmid is verified by streaking the cells on a selection plate with chloramphenicol. Transformation of the plasmid into Bacillus licheniformis (recipient) is achieved by conjugation with E. coli S17 (donor).

Homologous recombination occurs in two steps:

1. Single crossover: chromoslomal integration of the vector by homologous recombination is performed to maintain antibiotic resistance and because the plasmid cannot be replicated in Bacillus licheniformis.

2. Double crossover: elimination of the vector backbone from the chromosome by the scaB gene, which provides sensitivity towards sucrose.

3. Testing the Biosensor The biosensor is tested for the first time on beta-lactam antibiotic plates. The sensitivity of the whole-cell biosensor is then measured with different antibiotic concentrations, testing a range of beta-lactam antibiotics. In a final step, real wastewater samples are tested.

Parts: This page describes every basic and composite parts registered during the project.

Competent Bacillus licheniformis cells

To establish a whole cell biosensor using B. licheniformis we used several protocols to make competent B. lichenifimoris cells. As we were not able to verify the success of our efforts, we decided to still share all the protocols here so you may have a starting point for your own experiments. For this we are giving you references from which we took our protocols.

1. Kunst F, Rapoport G. Salt stress is an environmental signal affecting degradative enzyme synthesis in Bacillus subtilis. J Bacteriol. 1995;177:2403–2407

2. Z. Sun, A. Deng, T. Hu, J. Wu, Q. Sun, H. Bai, G. Zhang, and T. Wen, “A high-efficiency recombineering system with PCR-based ssDNA in Bacillus subtilis mediated by the native phage recombinase GP35,” Appl. Microbiol. Biotechnol., pp. 5151–5162, 2015.

Protocols PCR

As our goal is to establish a whole cell biosensor one of our main methods is the construction of and plasmid that encodes the signal cascade we want to archive.

For all PCRs we used the following pipetting scheme:

PCR pDM4 Vektor

Due to complications at the beginning of our experiments we settled on an annealing process with a temperature gradient. All of the bellow mentioned temperatures produced the needed plasmids at the according size.

PCR of sfGFP ́s

gDNA purification of B. licheniformis

1. A single colonie of B. licheniformis was picked and resupended in 10 µL of dH2O.

2. Sample is incubated at 95 °C for 15 min

3. PCR is used with the primers of the sequence you want to check for

Gibson assembly protocol

1 h incubation at 50 °C