Team:Paris Bettencourt/Project/Detect

   We developed a sensor to target antibiotic resistances in E.coli to test if a specific strain carries a certain antibiotic resistance gene. Our sensor system consists out of a phagemid with a CRISPR/Cas system and LacZ as a reporter under the control of a pRECA promoter (SOS response promoter).
If our CRISPR/Cas system can bind to the target (antibiotic resistance gene), the Cas9 generates at this specific target site a double strand break, which starts the expression of our reporter, as the promoter gets active at stress resulting from double strand breaks (Figure. 1). Having the system on a phagemid, the sensor system will spread all over a population, to get a clear color output if the target has been detected. Depending on target sequence the system carries, we can identify different antibiotic resistances in a strain. This is a novel approach of detecting genes in bacterial strains. We used E.coli and a M13 phagemid to target the kanamycin resistance gene.
This sensor is a proof of concept for a similar system in mycobacterium tuberculosis. Such a system could potentially be used to test if a patient has TB and what type of resistance genes the specific strain contains to adapt the patient’s drug treatment.

   Tuberculosis (TB) remains a major global health problem. While the treatment of this disease in countries with adequate medical care is fairly easy to detect and treat, it remains hard to treat and diagnose in poorer countries.
Up to now, a high quality lab that uses modern diagnostics is a prerequisite for early, rapid and accurate detection of TB. Therefore, diagnosis of TB and drug resistant TB remains a particular challenge for laboratory systems, especially in developing countries.
The lack of cheap, quick and accurate tests make it hard to control the Tuberculosis epidemic, which claims millions of lives every year in developing countries.
Setting up a cheap, fast and culture-based method could therefore decrease diagnostic time and facilitate patient treatment.
The most common method for diagnosing TB nowadays is sputum smear microscopy, in which bacteria are observed in sputum samples of patients under the microscope. However, this cannot be used to identify paucibacillary (containing just a few bacteria) or extrapulmonary (outside of the lungs) TB.

   To use our system, we need two strains, a phagemid producing strain and the target strain. The producer strain contains a helper plasmid, which produces the capsid proteins for the phage and the phagemid plasmid with the sensor elements (Figure 2), which is packed up into the M13 capsids (Figure 3). The helper plasmid encodes for everything of the phage but doesn’t contain the packaging sequence, while the phagemid plasmid only contains the packaging sequence but doesn’t code for anything else of the M13 phage. As M13 is a lysogenic phage, the phagemid particles are exported into the media, where they can be collected (Figure 4) and transferred to the target strain (Figure 5). The phagemid infects cells that are conjugating, as M13 needs a sex pili to attach to the cell and release the plasmid plasmid into the cell (Figure 6).