Bacterial microcompartments are primitive proteinaceous organelles synthesized by approximately 20 % of bacterial species whose genomes have been sequenced. Their main function is to improve the efficiency of metabolic reactions or to accumulate toxic metabolic byproducts. In general, depending on their function, they are divided into those that perform anabolic or catabolic reactions. Among the latter, the Pdu microcompartment is the most studied, as it is used in many biotechnological applications due to its modularity. In this master's thesis, we demonstrate a new application, more specifically, the encapsulation of a specific DNA fragment in the microcompartment that could be used to study protein-DNA interactions or as a scaffold for metabolic engineering. To facilitate isolation, we tagged the major subunit of the microcompartment PduA with the peptide tag Twin-Strep-tag, which allows affinity isolation of intact microcompartments. We show that the fusion protein PduD(1-18)-LacI-EGFP is efficiently encapsulated in microcompartments in vivo and remains functional after affinity isolation of the microcompartments. We prepared a system of two plasmids in Escherichia coli. The first plasmid carries selected components under an arabinose-inducible promoter, genes for: Yeast nuclease I-SceI, phage protein Gam, subunits of the Pdu microcompartment and the fusion protein PduD(1-18)-LacI-EGFP. On the second plasmid, there are one or two restriction sites for the yeast endonuclease I-SceI flanking the binding sites for LacI. Using high-throughput sequencing, we demonstrate that in the affinity-isolated microcompartments, the fragment carrying LacI binding sites is enriched relative to other regions of the genome, suggesting that this fragment was truly encapsulated. In addition, we show that we can encapsulate a DNA fragment carrying at least two different transcription factors in the microcompartments.
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