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Team:GeorgiaTech/Project
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Overall project
- Overuse of antibiotics in medical settings has created the problem of multidrug resistant bacteria, also called “super bugs,” which result from exchange of antibiotic resistance genes between bacteria by the process of horizontal gene transfer of plasmids. The experiment outlined in this proposal intends to engineer a unique method of “reverse” vaccination in which a plasmid (foreign DNA) containing an antibiotic resistance gene is specifically targeted and destroyed, rendering the bacteria susceptible to antibiotic treatment. The novel method we are developing utilizes a recently discover bacterial immune system called the CRISPR system (Clustered Regularly Interspaced Short Palindromic Repeats) which we intend to engineer onto a separate plasmid to inactivate the foreign DNA[1].
- The CRISPR system is a type of immunity predicted to be present in 40% of bacteria and about 90% of archaea [3]. It consists of a series of proteins, called Cas proteins, which are associated with a series of “spacers” separated by short palindromic sequences, that recognize foreign DNA (either plasmid or viral). If foreign DNA is recognized, the cell proceeses the invading DNA. If the DNA is not recognized and the cell survives the infection, it then “saves” pieces of the foreign DNA in subsequent spacers, to be used in future recognition of an attack.
- We will be creating a shuttle vector carrying the CRISPR system and use this plasmid-encoded CRISPR system to deliver this engineered immune system into bacteria. Our research aims to demonstrate that antibiotic resistance genes can be targeted with our engineered CRISPR plasmid in diverse bacterial species, rendering the bacteria more susceptible to antibiotic treatment. Specifically, a kanamycin (kanR) resistant plasmid will be targeted by our CRISPR plasmid that has been engineered to carry a kanR plasmid spacer. In addition, we intend to develop computer modeling approaches to be used for understanding CRISPR spacer uptake. Ultimately, new ways for targeting antibiotic resistance will be realized, helping to spark further research and innovation in this subject and CRISPR systems with real world applications.
References:
[1] Babu, M., N. Beloglazova, et al. A dual function of the CRISPR–Cas system in bacterial antivirus immunity and DNA repair (2011). Molecular Microbiology 79(2): 484-502.
[2] Horvath P., Barrangou R., CRISPR/Cas, the immune system of bacteria and archaea (2010). Science, 327(5962), 167-170.
[3] Barrangou, R., C. Fremaux, et al. CRISPR provides acquired resistance against viruses in prokaryotes (2007). Science, 315 (5819), 1709-1712
