Team:Tianjin/Results
Overview
In the Design section, we briefly introduced the experimental purpose and design ideas. In the Measurement section，we rigorously explain how we concretely realize these designs, and show the rationality of our process and the reliability of the results. In this section, we will correspond to it, summarize our final experimental data and results and make meaningful analysis to obtain new knowledge, re-evaluate the known and unknown factors in our experimental design. Finally feedback to the project to make it futher improved.
In this section，you can learn :
（1） We verify that the improved par--nfGFP has a shorter half-life.
（2） Through nucleic acid dyes, we observe the gradual disappearance of DNA in cells, thus verifying the formation of CREATE.
（3） By observing the rapid disappearance of nfGFP fluorescence signal in cells, it reflects the loss of gene expression ability of CREATE chromosome genome, thus confirming the formation of CREATE.
（4） By observing the growth status and colony number of the induced cells and normal cells on the solid medium, the results show a huge differences in the growth and reproduction capacity of the two groups of cells, which confirms the disappearance of chromosomes.
（5） Through flow cytometry analysis, we confirm the formation of CREATE by many parameters and signals and get the proportion of CREATE in the sample.
Due to the limitation of time, we have not complete all the experiments on exploring the features of CREATE, but the relevant experimental design has been determined.
You can click here to see the design
In the following subsections, we will analyze all the experimental results in detail.
Measurement of nfGFP half-life
We used changes in the nfGFP fluorescence signal to characterize chromosome degradation. In the beginning, we directly took the GFP part into practice, while later, it is observed that GFP has a relatively long half-life, which is not conducive to further measurement. So, we designed and constructed the nfGFP part with a shorter half-life to improve the feasibility.
Click here for detailed experimental design and discussion
Figure 1 The fluorescence signal degradation curve of nfGFP
After cessation of induction, the nfGFP signal decreased to the level when the expression was not induced, indicating that nfGFP was rapidly degraded in the cells after the fGFP gene was no longer expressed. The data show that the half-life of nfGFP is around 7.6h, which is significantly reduced compared to the half-life time of GFP on average, confirming the validity of our design.
Characterization of the formation of CREATE
Observe the disappearance of the nucleic acid dye
Nucleic acid dyes made it possible to visualize the degradation of chromosomes. Hoechest is a membrane-permeable nucleic acid dye that binds to DNA and generates a strong fluorescent signal. We stained the cells with the dye and photographed them under a fluorescent microscope to directly verify whether the cells' DNA was lost.
Click here for detailed experimental design and discussion
The following two figures show the changes in the control group (Saccharomyces cerevisiae 4742 nfGFP) after 18h of staining with Hoechst dye.
Figure 2 Hoechst control group 0h
Figure 3 Hoechst control group 18h
The control group showed an increase in the number of cells in the same field of view after 18 h, and the tiny dots in the cells with solid fluorescent signals due to Hoechst dye staining were always visible, which indicates that the control chromosomes were intact.
The following video shows the changes in the experimental group (Saccharomyces cerevisiae 4742 nfGFP with 7flip plasmid + Cre plasmid (cleavage system 2.0)) from 0 to 18h.
Video 1 Hoechst Experimental Group Video
In the video, it is observed that most of the fluorescence signal disappears, visualizing the process of degradation of brewer's yeast DNA, which indicates that the cleavage system takes some time to work and the efficiency of CREATE formation is not yet 100%.
nfGFP fluorescence signal attenuation
We use changes in the fluorescence signal of nfGFP to characterize chromosomal degradation. The nfGFP is a short half-life GFP that we designed, constructed, and experimentally validated.
More relevant information can be reached by clicking on the nfGFP part.
The following two figures show the changes in the control group (Saccharomyces cerevisiae 4742 nfGFP) in the nfGFP channel after 18h of induction.
Figure 4 nfGFP control group 0h
Figure 5 nfGFP control group 18h
It can be observed that the number of cells increased, and the overall average brightness was approximately the same, which indicates that the continuous production and degradation of nfGFP fluorescent protein in the control cells reached a balance, the nfGFP part works properly, and the cell growth condition was well.
The following video shows the changes in the experimental group (Saccharomyces cerevisiae 4742 nfGFP with 7flip plasmid + Cre plasmid (cleavage system 2.0)) from 0 to 18h.
Video 2 nfGFP experimental group vedio
Comparing to the control group, we observe that the nfGFP fluorescence signal turns significantly dimmer, as seen in this video. Reasons for this phenomenon we assumed are : 1. The cleavage system disrupts the chromosome, and thus cells can no longer express nfGFP, which indicates that our cleavage system works appropriately.
2. The half-life of the expressed nfGFP is short, and the degradation rate is fast, which also coincides with our data on nfGFP parts, indicating that our measurement of the half-life of nfGFP is plausible. In the meantime, since the mechanism of nfGFP rapid degradation involves organelles and enzymes, it also reflects that CREATE still has a complete cellular mechanism, organelles, and metabolic activities despite the loss of the genome. We also took pictures of the same field of view after removing the GFP channel, showing that although CREATE could no longer express nfGFP, the cell structure remained intact.
Figure 6 nfGFP GFP channel
Figure 7 nfGFP white light channel
For our thoughts on how to apply the CREATE, you can click here
Measurement of growth curve
We used the difference in the growth curves of the experimental (CREATE) and control (without the cleavage system) groups to show that our cleavage system worked successfully and obtained a chromosome-free eukaryotic CREATE that was completely different from normal cells.
According to other research, OD600 was positively correlated with cell concentration during the logarithmic growth period.
We used a UV spectrophotometer to measure the OD600 of the bacterial solution. By measuring the optical density of CREATE and normal cells, we obtained the growth curves of both. If it was indeed CREATE, it could not grow and reproduce, while the normal cells of the control could. We confirmed this difference and thus corroborated the formation of CREATE.
Figure 8 raw data of OD600
Figure 9 curve figure of OD600
From the figure, the experimental group that had their chromosomes cut did not significantly increase in yeast population compared to normal yeast cells, which coincides with our hypothesis that the CREATE cannot reproduce. After cutting, the growth curve of the colony in the experimental group should be below the normal yeast. The curve formed by the modified 7flip+cre cutting system is below the curve formed by the delta cutting system in the lower position, which shows that our improvement of the cleavage system is effective.
Flow cytometric analysis and sorting
The following two figures show the results of the flow cytometric analysis, the left graph is the control group, and the right is the experimental group. According to the analysis results, our highest chromosome-free eukaryotic cell CREATE formation rate reached more than 80%.
Figure 10 Flow cytometric control group/Figure 11 Flow cytometric experimental group
During the iteration of the project, we considered using the Saccharomyces cerevisiae SY14 strain, which has only one chromosome, as a chassis to make chromosome-free eukaryotic cells CREATE. We conjectured that SY14 might form chromosome-free cells more efficiently. We used flow cytometric analysis to compare the effect of the same cleavage system within different chassis strains simultaneously. To our surprise, the rate of chromosome-free formation was lower in strain SY14 than in strain 4742.
Figure 12 Comparison of strains in Flow cytometric
Point dilution plate and CFU dilution
we can observe the different growth status between experimental group and the control group based on the results of the point dilution plate.This indicates that the part is in working condition and indirectly support the formation of the chromosomal-free eukaryotic cell CREATE.
Figure 13 Different growth status between the experimental group and the control group
According to the number of colonies grown on the solid medium between the experimental group and the control group differed significantly. This indirectly support the formation of the chromosomal-free eukaryotic cell CREATE.
Figure 14 Different colony counts between the experimental group and the control group
The left is the experimental group (delta G) and the right is the control group (4741).The number of colonies after induction 48/120h is in the upper/lower half of the figure.
Exploration of the features of CREATE
To investigate whether cell morphology is affected when chromosomes are removed.
Figure 15 Microscopy of control group
Figure 16 Microscopy of experimental group
Compared to the control, no significant changes in cell morphology and size were observed in the experimental group, which indicates that even if the cells lose their chromosomes, the basic features of the cells will not alter within a short period.
Another result that can support this conclusion is that in experiments observe nfGFP signal when removing the GFP channel, and we can see that the morphological structure of cells without nfGFP fluorescence in vivo (CREATE) remains intact.
References
[1]John R. Houser,,Eintou Ford,,Sudeshna M. Chatterjea,... & Beverly Errede.(2012).An improved short‐lived fluorescent protein transcriptional reporter for Saccharomyces cerevisiae. Yeast(12), doi:10.1002/yea.2932.
[2]Hui Xu,,Mingzhe Han,,Shiyi Zhou,... & Ying-Jin Yuan.(2020).Chromosome drives via CRISPR-Cas9 in yeast. Nature Communications(1), doi:10.1038/s41467-020-18222-0.
[3]Fan Catherine,,Davison Paul A,,Habgood Robert,... & Huang Wei E.(2020).Chromosome-free bacterial cells are safe and programmable platforms for synthetic biology.. Proceedings of the National Academy of Sciences of the United States of America(12), doi:10.1073/pnas.1918859117.
[4](2018).Cellular Structures - Chromosomes; Investigators at Fudan University Describe Findings in Chromosomes (Creating a functional single-chromosome yeast). Science Letter(), doi:
[5]Shi Shuobo,,Liang Youyun,,Ang Ee Lui & Zhao Huimin.(2019).Delta Integration CRISPR-Cas (Di-CRISPR) in Saccharomyces cerevisiae.. Methods in molecular biology (Clifton, N.J.)(), doi:10.1007/978-1-4939-9142-6_6.