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From 2013.igem.org

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There are an estimated 10³¹ phage particles on earth, meaning that any bacterial process is constantly under the threat of infection. Industrial bacterial bioprocesses are no exception with phage infections frequently devastating bioreactor facilities, resulting in high economic cost. In fact, one of the main considerations in choosing the location of a bioreactor is the severity of environmental phage sources. Even in ideal locations, decontamination is frequently required and is the most substantial day-to-day financial burden. Engineering bacterial resistance to phage infection is a common scientific goal, however, but these attempts are usually undermined by the inherent diversity of phage. The bacterial immune system CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is one way in which bacteria naturally deal with phage infection in the environment and has become a powerful tool in genetic engineering with specificity at the single nucleotide level.

For the first time, we re-factored the CRISPR system down to its minimum components and demonstrated engineered specificity in E. coli. We analyzed all available sequence information from two common phage families that infect E. coli and designed the most broadly neutralizing systems possible. The work provides a proof-of-concept experiment for engineering bioreactor immunity and provides all the sufficient modules to facilitate future engineering of CRISPR in bacteria. Moreover, having these working components in the biobrick registry is incredibly exciting as it’s tractability will endlessly expand the engineering potential of the iGEM community going forward.

An example of one such industrial process that is susceptible to phage-mediated collapse is the fermentative production of yogurt. With the amount of work that the iGEM community has put into the production of compounds such as Vanillin and Caffeine, we envision the next generation microbes in yogurt fermentation being able produce these products in vivo. Consolidating these, however, might not be efficient because while many people may want vanilla flavored yogurt, there might only be a subset of those who want the caffeine boost. This got us thinking about engineering CRISPR in a way that allows population control in a bioreactor. For example, there could be a vanilla producing bacterial strain that is immune to all the known environmental phage while a caffeine producing strain is also immune, but selectively susceptible to a rare phage. In a co-culture fermentation, which is the situation in yogurt production, the system could be phage resistant but the targeted addition of a rare phage could knock down a subset of the population and effectively decaffeinate the fermentation. We worked on biosynthetic pathways such as those producing vanillin and caffeine to further part characterization in addition to the modeling and validation of parts in our hypothetical system. A proof-of-concept experiment showing targeted population control using CRISPR in E. coli is underway and will be presented in Toronto.