Team:NPU-China/Future work
This year we designed the genome sequence of MitoCRAFT and initially verified the de novo synthesis of this genomic
DNA. Due to the cumbersome and synthetic difficulty of the synthesis of this genome, we have not yet converted and
functionally verified MitoCRAFT, the real application of MitoCRAFT still remaining a long way to go. Although this
year's competition is coming to an end, the pace of our exploration of the limits of life shall never stop. We have
been deeply enchanted by the MitoCRAFT project and pledge to continue to complete the testing and optimization of
MitoCRAFT after the competition.
The following are our detailed plans:
1. Transformation of the MitoCRAFT genome
The transformation of mitochondrial DNA has been a focused scientific problem in academic research. Our consultant,
Professor Gu Zhenglong from Cornell University, has also been exploring efficient methods of mitochondrial genome
delivery. In the light with their current experimental results, it can be known that they have achieved efficient
transformation of the mitochondrial large genome (150 kbp) based on the gene gun method, which is indeed good news
for us. Prof. Gu has agreed with us that after the completion of our genome sequencing, he will assist us in
transforming the MitoCRAFT genome.
2. Functional verification of MitoZero
According to our goal, MitoZero is supposed to have normal transcriptional translation function and maintain a
certain aerobic respiration. We will use the characteristics of S. cerevisiae Rho0 cells (missing mtDNA) that they
cannot grow in a medium containing a non-reducing carbon source to screen for Saccharomyces cerevisiae containing
MitoCRAFT that can maintain aerobic respiration.
If we can access effective cloning, we will draw on Dr. Xue Xiaoli, who have successfully achieved the fusion of 16
chromosome genomes of Saccharomyces cerevisiae into one this year [1], to measure the changes in the relevant
metabolomics and transcriptomics parameters of this S. cerevisiae. Instead, if we fail, we will use engineering
ideas to test each of the major processes. For instance, we will use RT-PCR to verify the transcriptional function
of mitochondria, identifying whether the transcription of each functional gene is normal and apply Westen-blot to
verify whether the subunit related to aerobic respiration is translated, then the MitoZero genome will be optimized
on the basis of the test results.
3. Control test verification of gene region knockout
When we introduced our project to other teams, they all felt that our design was too radical, especially the
deletion of mitochondrial introns that may bring about the failure of these genes. Considering the possible
"failures", we have also designed two control test genomes where the intron and intergenic regions were separately
simplified.
Diagram of the separate deletion test of intergenic regions:
Figure 1.Control test genomic structure map of intergenic regions
The above PCR fragments were amplified from the wild-type mitochondrial genome by PCR and the test genomes of the
deleted intergenic regions were spliced based on our current genomic splicing technique and experience. In order to
verify the function of control test genomes as soon as possible, we first tested the separate deletion of Delete1.
Figure 2. Diagram of the separate deletion test of introns:
We plan to do a separate deletion test for the COX1 gene intron. First, by reference to the separate deletion test
method of the intergenic region, the rest part except the COX gene in the figure above will be amplified, then we
will test the existing COX gene CDS after assembly.
If you also crave to build MitoCRAFT together with us after seeing our project, please do not hesitate to contact
us.
Reference
[1]. Fang K, Chen X, Li X, et al. Super-resolution Imaging of Individual Human Subchromosomal Regions in Situ
Reveals Nanoscopic Building Blocks of Higher-Order Structure[J]. ACS nano, 2018, 12(5): 4909-4918.
