Team:NPU-China/Mini mtDNA Design
MitoCRAFT is a sequence-simplified Saccharomyces cerevisiae mitochondrial chassis (approximately 39Kbp) consisting
of two parts. One is a pure chassis that provides a basic transcription and translation system and maintains aerobic
respiration, which we call MitoZero; the other is an extension that extends the functionality to create a
diversified toolbox, which we call CRAFT Plus.
1.Design of MitoZero
The gene-scale diversity of the mitochondrial genomes of S. cerevisiae (from a minimum of about 20 kbp to a maximum
of around 130 kbp) and its significant linear positive correlation with genomic scale and intergenic regions have
drawn our attention. We wondered whether they could be “restored” by simplifying redundant sequences of genes and
complicated gene regulatory sequences, creating a relatively primitive S. cerevisiae mitochondrial chassis,
MitoZero, which provides the basis for a further extended and more complex manual mitochondrial design.
The goal of the design of MitoZero: On the basis of guaranteeing the basic mitochondrial function, the sequence
ought to be simplest and has a relatively simple genetic structure, which facilitates the subsequent complex design.
The scheme of the design of MitoZer: Based on the bioinformatics analysis model, the functionality and relative
conservation degree of each part of the genome will be determined (by reference to generalized and narrowly defined
Saccharomyces cerevisiae) and the non-conserved part of the intergenic region which accounts for a large proportion
of the original mitochondrial genome and the intron regulatory system that is not conserved in the genome will be
simplified.
1.1 Simplification of non-conserved intergenic regions and reduction of genome size
In the course of the evolution of species, the intergenic regions also altered with the change in the relative
position order of the gene modules. Generally speaking, intergenic sequences are difficult to survive under such
changes if they need to perform important biological functions. Therefore, in contrast, the intergenic regions
within the syntenic orthologous blocks, the gene regions in line with the relative position order, are more crucial,
while the intergenic regions between the blocks can be simplified to some extent.
Utilizing the Gen Order Conservation (GOC) calculation model and the Branch-specific Gene Order Loss (bsGOL)
calculation model (see Model), we obtained the specific positions of the 7 blocks of the genome. We retained the
sequence of 100 bp upstream and downstream of each block as sequence buffers and deleted the redundant sequences of
the remaining intergenic regions, totaling 29,301 bp.
1.2 Simplification of non-conserved introns as well as construction of a more primitive transcription and
translation regulation system with the reduction of genome size
In the process of conservation analysis of wild-type mitochondrial genome introns, we found that intron sequences
are much less conserved than exons (only one is conserved), thus being able to be simplified to create a relatively
simple functional gene transcription and translational regulation system. We first removed all non-conserved
introns. Considering that only one intron had no corresponding shear control system, we also removed it. The
simplified introns have a total of 17,512 bp.
Through the simplification of these two aspects, we have created the theoretically primary minimal Saccharomyces
cerevisiae mitochondrial chassis - MitoZero, the sequence of which has been reduced from the original 85,779 bp to
38,966 bp, less than half of the original length.
Figure 1.MitoCRTFT gene map without GPF
2. Design of CRAFT Plus
Saccharomyces cerevisiae mitochondria have correspondingly independent functional expansion capability due to their
relatively independent transcription and translation system. Compared with the original genome of up to 86kbp, the
simplified genome scale has been remarkably reduced. In theory, it is easier to perform molecular cloning
operations, elevating the degree of easy “insertion and removal” of its function expansion.
We designed a GFP-based reporter gene module to verify the function expansion capability of MitoZero.
The GFP module is a simple promoter-CDS-terminator structure whose promoter sequence is a strong one from the
original mitochondria, 5'-ATATAAGTA-3', and similarly, the terminator sequence is 5'-AATAATATTCTT- 3'. Since the
mitochondrial codon table differs from the nuclear genome, we have conducted targeted codon optimization.
The codon optimization table:
（Note: This codon is MitoCRAFT Coden Usage Table based on the Saccharomyces cerevisiae mitochondrial codon table.
Copyright belongs to the original author.）
We have provided the simplest example for followers through our design.
Figure 2.Complete MitoCRTFT gene map containing GPF
