In 2019 an estimated 1.2 million people died of antibiotic resistant pathogens
1. That is more deaths than caused by HIV
2.
The World Health Organisation assesses the situation as such that “A post-antibiotic era – in which common infections and minor injuries can kill – far from being an apocalyptic fantasy, is instead a very real possibility for the 21st Century.”
3
While many other teams have worked and are still working on new treatments to fight those resistant germs (as
iGEM Hamburg 2017) we want to speed up the diagnosis of bacterial pathogens. Far too often antibiotics are prescribed without a clear diagnosis of what caused the sickness and if possible resistances exist, as happened to one of our team members this year. That is often necessary as antibiograms are dependent on bacterial growth and often take days to give a result, time that doctors cannot waste in treating a patient. This is our motivation to work on
SPEAR -
Sensing
Pathogens and
Emerging
Antibiotic
Resistances.
We aim to improve the detection of bacteria and possible resistance factors by directly recognizing the corresponding RNA. By being independent of bacterial growth we reduce the time for diagnosis. To achieve this we use split ribozymes
4 developed by Lauren Gambill et al. they are a very useful tool in detecting long RNAs.
Ribozymes are catalytically active RNAs. Which means they can cut themselves out of an mRNA for example. Many ribozymes have been used in iGEM for different applications before. However they normally can not be utilized to directly detect RNAss. Gambill et al. were the first to split the ribozyme into two parts and add so-called guide RNAs(gRNAs) in between. The gRNAs are chosen to be complementary to any target RNA, in our case bacterial RNA or resistance gene mRNAs. By binding to the target mRNA the gRNAs restore the catalytic function of the ribozyme leading to the expression of a reporter gene. Gambill et. al analyzed the most potent split-ribozymes, optimizing gRNA length and splitting point in the ribozyme. Additionally they built different split-ribozyme constructs at different positions in the mRNA, either inside the coding sequence (CDS) or between ribosome binding site (RBS) and CDS. We decided to use the latter design, as it is more modular and therefore better to use in the iGEM context. It also enabled us to test different reporters downstream of the ribozyme to find the best one for our application.
We then designed our own gRNAs targeting bacterial RNA hcat, a stably expressed RNA of a putative 3-phenylpropionate transporter in
Escherichia coli , resistance mRNA of kanamycin and mRNA of GFP to easily assess levels of the target mRNA. We combined these split ribozymes with different reporters: enzymes (horseradish peroxidase (HRP), chromoproteins (eforRed, Bba_K592012) and fluorescent proteins (GFP). That way we could test which reporter is the best for our application. In the end we plan to develop a point-of care diagnostic device enabling the easy and quick detection of specific RNAs. Check out our
Results page to see how it worked out!
