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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 and phage infections frequently devastate bioreactor facilities, resulting in high economic cost. In fact, one of the main considerations in choosing the location of a bioreactor is the 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, 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 its tractability will endlessly expand the engineering potential of the iGEM community going forward.