Team:Warsaw/Project/conjugation

Conjugation with mitochondria
Conjugation with mitochondria is one of the possible applications our BacInVader system. This is based on publications showing that E.coli has the potential ability to conjugate with eucaryotic mitochondira in vitro[22].

Theoretical basis of mitochondrial transformation
Electron transport chain functionality can be impaired by a variety of defects on the DNA level: not only point mutation and local deletions in genes encoding proteins of the chain, but also deletions of larger fragments or even the whole mitochondrial genome (mtDNA). Normal mitochondrion contains from 2 to 10 copies of mtDNA. Defects in mitochondrial DNA result in severe medical disorders which are especially evident in tissues which require large amounts of energy (such as nervous and muscle tissue). Effects of these impairments are neuro and myopathies [20]. The standard therapy based on bypassing interrupted pathways does not apply under these circumstances. The administration of enzyme cofactors and other metabolically active substances is not largely effective and may be applied only as supplementary therapy. We are forced to seek novel treatment strategies to cure patients suffering from mitochondrial disorders [7]. Currently the most commonly used method of mitochondrial transformation is the "gene gun" approach which is based on the delivery of metal beads coated with DNA into the cell. This method is mostly limited to plants and fungi and has low therapeutic potential [7]. Current mammalian mitochondrial transformation techniques like electroporation also have serious limitations. Whole process takes place ex vivo and causes irreversible damage to the mitochondrion which makes its efficient reintroduction into the cell almost impossible. The conjugation between bacteria and mitochondria is free from these side effects. Moreover, this phenomenon has been preformed in vitro.[22]. More recently a conjugation between two strains of bacteria within the cytoplasm of a mammalian cell has been observed. These findings suggest that conjugation between a bacterium and a mitochondrion is feasible [10].

Details
To locate the copy of the mitochondrial genome into bacterial cell the pBACrNESd plasmid will be used. It is based upon the Bacterial Artificial Chromosome. Replication origin (ori R6K), kanamycin resistance gen (kanR) and replication complex genes (repE, parA, parC) from pBACrNESd plasmid will be fused with origin of transfer (oriTF) from a natural conjugative F plasmid. Unique restriction site will be introduced into the mitochondrial genome by PCR reaction and the described construct will be inserted into mitochondrion. To enable conjugation proteins encoded on RP4 plasmid (Tc, MuKm, Tn7) will be placed in the cytoplasmic operon. These genes are responsible for pillus formation and transport of a single stranded DNA. The cell with this system will conjugate with any structure surrounded by a lipid bilayer. We will prepare mitochondrial DNA deficient cells using a previously described procedure [2]. The occurrence of conjugation will be confirmed by PCR reaction specific for sequences added to mitochondrial DNA. The origin of the DNA will be determined by analysis of restriction patterns. The expression of mitochondrial genes will be confirmed by semi-quantitative PCR [2].