Team:Paris Bettencourt/Project/Detect

   We developed a sensor to detect whether a particular bacterial strain carries a specific antibiotic resistance gene. Our sensor system consists of E.coli lab strain and of a synthetic 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 will induce the expression of our double-strand break SOS sensor (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, Step i), which is packed up into the M13 capsids (Step ii). 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 (Step iii) and transferred to the target strain (Step iv). 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 (Step v).

Step i: Producer strain containing a helper plasmid for M13 and the phagemid plasmid with the sensor elements.

Step iii: Collection of phagemids in the media.

Step v: Release of the phagemid plasmid into the cell.

Step vii: Fusion of gRNA with Cas9.

Step ix: Site-specific cut of Cas9 in the kanamycin resistance gene. Due to double strand break activation of the pRECA SOS response promoter.

Step xi: Cells turn blue.

   The mechanism we are using for our sensor is based on the CRISPR/Cas System. The CRISPR/Cas genome editing method, which recently became very popular, is derived from the bacterial “immune system”. CRISPRs are Clustered Regularly Interspaced Short Palindromic Repeats that are part of the bacterial genome. These loci contain multiple short repeats. In between the repeats are so called spacers that are sequences derived from extrachromosomal DNA, e.g. from invading viruses or plasmids (Figure 3). Within bacteria, those CRISPRs are naturally used to detect and destroy foreign DNA that is saved in the spacers.

In total the CRISPRs and the spacers form a so-called CRISPR array that is transcribed as one unit. CRISPR associated proteins (Cas proteins) are involved in further processing and action steps of the CRISPR system. There are many different regulation systems, here we describe the Cas9 system that will be used for our purposes (Figure 3).

The transcribed mRNA is processed into so called crRNA (CRISPR RNA), which includes the spacer sequence of foreign DNA. Together with a transactingCRISPR RNA (tracrRNA), the crRNA forms a duplex that is cleaved by RNaseII. The resulting hybrid serves as guide for the Cas9 protein that generates double strand breaks at the position the RNAs guide it to (Figure 3). By this double strand break the invading DNA is destroyed.

RecA promoter

   Our reporter system consists out of the RecA promoter and LacZ. Originally, the RecA promoter of Escherichia coli has its main role in the activation of the SOS repair system. It is regulated by the LexA repressor, which binds to the SOS box sequence of the promoter. DNA damage leads to an inducing signal, which then activates the RecA protein. In the SOS response the LexA repressor is cleaved by the RecA protein, so the full RecA expression can be reached. The RecA protein can then repair both single stranded and double stranded DNA breaks.

In our project RecA promoters used to detect double stranded DNA breaks in the region of antibiotic resistance genes caused by the CIRSPR/Cas system. To get the desired activation of the promoter, it is important to assure that no other stressing agents might activate the RecA promoter. Those agents could be UV light, X-ray, ionizing radiation and different types of DNA breaking compounds.

   Up to this point we cloned each of our parts into BioBrick vectors. We cloned the gRNA anti-Kan into pSB1C3 and pSB1A3. We used for this the assembly standard BioBrick cloning as well the new proposed Assembly Standard.. Both assemblies were successful and proved by colony PCR as well as by sequencing. We also used both standards for cloning the crRNA.. Also this is verified by colony PCR and by sequencing. For the cloning of the tracrRNA-CAS9 part we used standard BioBrick cloning, inserted into pSB1A3 and sequence verified it. We achieved the same for the pRecA-LacZ part (pSB1C3 and pSB1A3 backbones) that was submitted to the parts registry. As soon as possible we will start the characterization of our parts. For the characterization of the tracrRNA-Cas9, the gRNA anti-KAN and the crRNA anti-KAN we plan a killing assay.