Team:Strasbourg/Our scientific strategy

iGEM

Many detection gadgets are based on antibodies which are expensive because of the use of animal for their production and mass spectrometry for their quality analysis. iGEM Strasbourg team is working on the development of a new detection system, which does not require antibodies. It should be fast and portable for easy on-site use, as well as versatile in order to be adaptable to any molecule. Our detection kit is based on a triple hybrid system (BH3) implemented in E. coli. Flexibility will be provided thanks to an aptazyme that can be interchanged for the detection of a specific ligand. The aptazyme switch is an RNA molecule that has an aptamer domain and a ribozyme domain, a breakthrough in molecular biology. Two different molecular platforms will be tested.

Description BH3 Project

iGEM Strasbourg team adapted the existing aptazymes to BH3 platform developed by Berry et al. and published in 2018 [1] by adding MS2 and PP7 stem-loops to its 5' and 3' extremities, respectively.

This bacterial three-hybrid construct is composed of two fusion proteins. A DNA-bound protein is fused to the coat protein of bacteriophage MS2 (RNA-binding moiety), and used to recruit the aptazyme upstream of a beta-galactosidase promoter. On the other side, the aptazyme interacts with a RNA-binding PP7 coat protein that is fused to a subunit of E. coli RNA polymerase (RNAP). This fusion stabilizes the constant binding of RNAP to the promoter, thereby activating the transcription of a reporter gene (here ß-galactosidase) (Fig.1).

Figure 1: iGEM Strasbourg triple hybrid system

In addition, we tried two different aptazymes to select the better conditions to investigate our detection system: an aptazyme switch-on and an aptazyme switch-off (Fig.2). This allowed us to test two variants of the BH3 system. In the case of switch-on the presence of the target ligand activate apatazyme self-cleavage, which prevents RNAP recruitment on the beta-galactosidase promoter and leads to the prevention of the reporter gene transcription. In the case off switch off, the presence of ligand inhibit Aptazyme calalytic activity and the reporter gene is activated.

Figure 2: The switch-off (e.g. theophylline) and switch-on (e.g. guanosine) aptazymes

Description LexA Project

The mechanism is based on a LexA repressor system [2]. It involves the detection of a ligand by an aptazyme that reverse this system by activating the transcription of a reporter gene (Fig.3).

Indeed, LexA (22.7 kDa) is a transcriptional repressor involved in SOS-system (a DNA damage response) by its binding with a promoter sequence of SOS-response regulatory genes. This leads to the expression of genes involved in DNA repair and survival of the bacterial cell. It is regulated by LexA and RecA proteins and stimulates the autocatalytic process of lexA (an intramolecular cleavage of Ala84-Gly85 bond in LexA repressor).

Figure 3: iGEM Strasbourg LexA system

The detection process involves two fusions proteins, which are two versions of chimeric LexA dimer: LexA with wild type DNA binding domain fused to the PP7 coat protein and LexA with the mutated version of DNA binding domain fused to the MS2 coat protein. Respectively, these two hybrid proteins specifically recognize the mutated LexA operator sequence, which contains a wild-type half-site (CTGT) and a mutated operator half-site (CCGT) described by Dimitrova et al 1998 study.

The aptazyme contains an aptamer domain-specific to the tested ligand and a self-cleaving ribozyme and it interacts with bacterial three-hybrid construct due to added MS2 and PP7 RNA stem-loops.

When the target ligand is in the vicinity of the three-hybrid system, the aptazyme interacts with it (ligand) and can cleave itself (by the reject of the wt-lexA-PP7 hybrid protein). This cleavage leads to preventing the activity of the repressor LexA. It allows the activation of the ß-galactosidase reporter gene transcription process thanks to the recruitment of the RNA polymerase

The possibility to choose between a switch-on or a switch-off aptazyme is also given here.

References

  1. Berry, K. E., Hochschild, A. 2018. A bacterial three-hybrid assay detects Escherichia coli Hfq-sRNA interactions in vivo. Nucleic Acids Research 46.
  2. Dimitrova, M., Younès-Cauet, G., Oertel-Buchheit, P., Porte, D., Schnarr, M., and Granger-Schnarr, M. 1998. A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli. Molecular and General Genetics MGG 257, 205–212.
  3. Daines, D. A., Granger-Schnarr, M., Dimitrova, M., Silver, R. P. 2002. Use of LexA-based system to identify protein-protein interactions in vivo. Methods Enzymol 358:153–161.
  4. Daines, D. A., Silver, R. P. 2000. Evidence for multimerization of Neu proteins involved in polysialic acid synthesis in Escherichia coli K1 using improved LexA-based vectors. J Bacteriol 182:5267–5270.
  5. Felletti, M., Stifel, J., Wurmthaler, L.A., Geiger, S., and Hartig, J.S. 2016. Twister ribozymes as highly versatile expression platforms for artificial riboswitches. Nature Communications 7.
  6. Stifel, J., Spöring, M., Hartig, J.S. 2019. Expanding the toolbox of synthetic riboswitches with guanine-dependent aptazymes. Synthetic Biology 4.