Team:Tianjin/Demonstrate
Part1. Characterization of Chromosome Stabilization Element Group
Unlike most iGEM teams that use plasmid construction, most of our manipulations are performed on the yeast genome, we assemble and build our parts with the centromeres of Saccharomyces cerevisiae or Yarrowia lipolyticae.
In order to integrate the centromere into another cell, we have taken two approaches.
1. Adding centromere of Saccharomyces cerevisiae chromosome XV to Schizosaccharomyces pombe chromosome III
We first tested the related functions of chromosomal stabilization element group in Schizosaccharomyces pombe, which is far away from Saccharomyces cerevisiae. We chose a location far away from the peripheral heterochromatin of the original cells. Considering the need for the cell itself to propagate, we did not destroy its peripheral heterochromatin structure and avoided the kinetochore binding region
Figure 1. Insertion position of centromere
Figure 2. Sequencing results of transformant centromere fragments (the blue part is the sequencing result)
2. Add centromere of the Yarrowia lipolytica E chromosome to the Saccharomyces cerevisiae V chromosome
This is an another attempt by us to directly insert new centromeres into the peripheral heterochromatin region. The purpose of this is to ensure that this is in the fusion system without affecting the original cells as much as possible. The location of the chromosomal binding site of the chromosome and the dividing chromosome are consistent with those before fusion.
There is currently no literature on centromere epigenetics that clearly indicates the consequences of doing so, and our follow-up test results may provide some reference for this research.
Figure 3. Insertion position of centromere
Figure 4. Growth status of transformed cells
Figure 5. The morphology of transformed cells
Figure 6. Growth curve of transformants versus original cellsa
(a.Saccharomyces cerevisiae b.Saccharomyces cerevisiae inserted into the Yarrowia lipolytica centromere c.Saccharomyces cerevisiae inserted the centromere of Yarrowia lipolytica)
Figure 7. Phenotypic analysis of three srtins
By comparing the transformants with the original cells, we find that they apparently do not change much in morphology and growth pattern, and the metabolic pathways on this chromosome that can produce lycopene can still be expressed normally.
However, we still find that the growth of the transformants is slower. This may be due to the fact that multiple centromeres on the same chromosome work together during the cleavage phase. Even if they are closely spaced, there may be a phenomenon in which the entire chromosome is broken or cannot be separated from the middle. This is also covered in our modeling, click here for more information.
Figure 8. PCR validation map of transformants (D and E are the two transformants we verified. From left to right are the connection points of L-his, his-cen and cen-R)
Figure 9. Sequencing results of transformant centromere fragments (No. 4 and No. 12)
This shows that the centromere fragment we integrated into S. cerevisiae is correct.
3. Directly replace the original centromere of S. cerevisiae with the centromere of Y. lipolytica
After obtaining the results of the above two experiments and conducting relevant analysis, we have new ideas. Comparing the Saccharomyces cerevisiae with the centromeres of the Yarrowia lipolytica, we can see that there is not much difference between them overall. They both have point centromere, and the two yeasts are closely related, and there is no clear evidence that Saccharomyces cerevisiae cannot recognize the centromere of the Yarrowia lipolytica. So after replacement, we will be able to obtain a Saccharomyces cerevisiae that uses the Yarrowia lipolytica centromere to direct chromosome-dissociation-related behavior.
Figure 10. Location of the centromere replaced (the position between the left and right homology arms)
Figure 11. The morphology of transformed cells (on the left is Saccharomyces cerevisiae, on the right is Saccharomyces cerevisiae inserted the centromere of Yarrowia lipolytica)
The growth curve and dilution of yeast are shown in Figure 6 and Figure 7.
From the growth status of Saccharomyces cerevisiae, the morphology and growth of the transformants and the manner of division did not change much, and the metabolic pathway capable of producing lycopene on this chromosome could still be expressed normally. It shows that the whole cell can still grow normally after replacing the centromere.
Figure 12. PCR validation map of transformants (the brighter strip in the figure is the fragment where the centromere is located)
Figure 13. Sequencing results of transformant-related fragments
This shows that the centromere fragment we integrated into Saccharomyces cerevisiae is correct.
We also compared the lycopene production (by comparing the color) of yeast inserted with centromere, yeast replaced with centromere and original Saccharomyces cerevisiae. It was apparent by visual observation that the yield of Saccharomyces cerevisiae with an additional heterologous centromere was significantly lower than the other two, and the Saccharomyces cerevisiae replacing the centromere was similar to the original yeast.
By comparing the data of three kinds of Saccharomyces cerevisiae, such as morphology, growth curve and apparent yield of lycopene, we can draw some preliminary conclusions:
1. Saccharomyces cerevisiae can recognize the centromere of Yarrowia lipolytica, and the growth, reproduction and related functions of the whole cell are not affected after replacement.
2. The function of point centromere and regional centromere is orthogonal.
3. When there are two functional centromeres on a chromosome, it will cause problems in the recognition of related proteins during the cleavage stage, which may affect the division process (and may even lead to abnormal chromosome structure), which will eventually affect its growth and reproduction.
In general, centromere is part of the chromosomal stabilization element group and in the process of constructing it, we found that it can have a certain impact on the growth and reproduction of the cell itself.This is inconsistent with the information we had previously expected from the literature. In order to find out the reasons behind, we are going to conduct bioinformatics analysis of the centromeres of these three yeasts.
Part2. Characterization of Chromosome Transfer Elements
1. mRFP
We transformed mRFP into the genome of Saccharomyces cerevisiae and expressed it, and photographed it with a fluorescence microscope before and after fusion to confirm whether this chromosome has metastasized.
Figure 14. Photos before and after fluorescence excitation under fluorescence microscope
If red fluorescence is detected from the fusion system after red light excitation, then the entire chromosome can be proved to have metastasized without being digested.
2. Single base editor based on crispr-dcas9
We obtained the plasmid containing the fusion protein directly from the laboratory, and verified it simply by PCR.
Figure 15. Plasmid profile
Reference data for the efficiency of fusion proteins are derived from the literature.
2.1 Construction of gRNA
In order to ensure the randomness and orthogonality of gRNA, we designed a small program, which can generate multiple gRNA sequences that meet the individual needs of users on the premise of meeting the randomness requirements.
According to the program we designed, we generated several sequences that meet the requirements, and chose one of them for our experiment.
We constructed fragments containing gRNA that we needed by using the method of PCR and integrated them into the relevant fragments in the genome.
2.2 Construction of protospacer
We linked the sequence we need to edit to the back of the tag on the plasmid by enzyme cutting and enzyme linking, and obtained a related fragment which can be integrated into the genome by PCR.
Part3. Characterization of Fusion Results
When we have confirmed that the centromere has been constructed, we can continue to experiment to confirm the effectiveness of this set of chromosomal stabilization element group. Because there is no way to introduce a whole heterochromosome into yeast now, we choose the old strategy——fusion, and in order to ensure the correctness of the growing fusant, we culture the fusant on the double screening medium. The method we used is pegc mediated protoplast fusion.
1. Fusion of Saccharomyces cerevisiae and Yarrowia lipolytica
Figure 16. Morphological comparison between fusant and original Saccharomyces cerevisiae and Yarrowia lipolytica
Figure 17. Morphological comparison between fusant and original Saccharomyces cerevisiae and Yarrowia lipolytica
We can clearly find that there are considerable individual differences between the fusants (in terms of cell morphology, colony size, color, etc.). This is due to the nature of the fusion itself, and the genetic material retained in each fusant is different.
The initial fusant is unstable and in a "wobble" state between the two kinds of yeast, returning to its original state after several generations. This phenomenon is uncontrollable in the traditional fusion, and the result is also random and greatly disturbed by the external conditions.
As we expected, after adding chromosomal stabilization element group to one cell, the unstable state will gradually converge to a "metastable state" with the increase of time and the number of generations. That is, the chromosome with the other kind of centromere will remain and the other heterochromosomes will disappear. The time needed for the complete disappearance of chromosomes may be determined by the nature and affinity of the two cells.
Figure 18. PCR verification results of fusant chromosomes
In order to characterize the internal chromosome number of fusant, we have carried out PCR verification (including several random Saccharomyces cerevisiae chromosomes and some regions at both ends of all the chromosomes of Yarrowia lipolytica).
The above figure is an example of our high-throughput test. Three chromosomes of Saccharomyces cerevisiae and Yarrowia lipolytica are selected for verification in a group of six test points.
According to the current experimental results we had, most of our fusants prefer Saccharomyces cerevisiae. Most of the PCR results of the Yarrowia lipolytica chromosomes in the fusants are dim or have no corresponding bands, which may be related to the overall culture conditions and the usage of the related reagents in the fusion process determined by Saccharomyces cerevisiae. However, these Saccharomyces cerevisiae are not pure Saccharomyces cerevisiae, but the Saccharomyces cerevisiae that contains partial Yarrowia lipolytica gene, they get the tag of the other original cell in the process of "swing".
2. Fusion of Saccharomyces cerevisiae and Schizosaccharomyces pombe with new centromere
Figure 19. Morphological comparison of fusant with original Saccharomyces cerevisiae and Schizosaccharomyces pombe
After several high-throughput screenings, we can see that at the beginning of fusion, the related parts of most of the chromosomes still exist, but as time goes on and the increase of the number of generations, the extra transferred chromosomes begin to disappear gradually.
After analysis, we get some conclusions:
Some genes of one kind of yeast in the fusant may be randomly integrated during the digestion of these chromosomes.
For the process of chromosome transfer by fusion, it is far from enough to rely on a centromere which can guide chromosome separation in the mitosis stage. According to the results we have obtained, the original assumption is that the chromosome with centromere will not be broken down and digested after being transferred to other cells, which is not fully true. Therefore, we may need to add new telomeres or optimize the sequence to prevent recognition by nuclease.