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Single cells have precise spatial and temporal control over gene expression and exhibit a sensitive regulatory architecture to be able to survive in a variety of different environments. Starting from the blueprint genome, the complete orchestration of interactions between proteins, nucleic acids, lipids, etc. is poorly understood in even the simplest of bacteria. Major thrusts to create a minimal cell have resulted in successful synthesis of minimal genomes consisting of only ~400 genes, giving scientists a reduced system to try to understand basic principles behind the construction of life. Currently only ~100 genes within the minimal genome have not been characterized, leaving only 25% of the genome unknown. In this work, we describe the construction of a genome engineering tool, CRISPRi, to modulate the expression of a particular gene of interest.
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Design
Inducible gene expression of CRISPRi is modulated by a theophylline dependent riboswitch
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ConstructionThe plasmid is constructed through E. coli assembly with the riboswitch directly upstream of CRISPRi
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ExpressionThe plasmid is transfected into syn3.0 for expression of CRISPRi and attenuation of specific genes of interest
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