Team:Tianjin/Measurement

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Overview


In the design section, we briefly introduce the purpose and design ideas of our experiments. In this section, we will correspond to it, rigorously explain how we concretely realize these designs, and show the reasonability of our process and the reliability of the results.
In this section,you can see:
(1) How we measure the degradation rate of the improved part--nfGFP and verify the shortening of its half-life.
(2) How we use fluorescence microscope to observe the disappearance of dye-stained DNA to verify the formation of CREATE.
(3) How we observe the disappearance of nfGFP fluorescence to prove that CREATEs are generated.
(4) How we measure the growth curve of the experimental group and normal cells to show the difference between the growth and reproduction ability of the two groups of cells, thus proving the formation of CREATE.
(5) How we set parameters and sort CREATEs through the flow cytometer and get the formation rate of CREATEs.
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 learn more

In the following subsections, we will discuss the above experiments in detail.

Measurement of the half-life of nfGFP

Experimental purpose


We used changes in the nfGFP fluorescence signal to characterize the degradation of chromosomes.
In the beginning, we directly took the GFP part into practice, but it is observed that GFP has a long half-life, which is not convenient to further measurement. So, we designed and constructed the nfGFP part(new GFP which degrades fast) with a shorter half-life to improve the feasibility.

The characterization plasmid of nfGFP


Ubiquitin is a small protein found in all eukaryotes whose primary function is to label a protein to be quickly recognized and degraded by the proteasome in the cell. We added a ubiquitin tag before the GFP structural gene, predicting that this would result in faster recognition and degradation of the expressed ubiquitin-tagged-GFP fusion protein by the ubiquitin degradation mechanism [1]. To test our hypothesis, we designed the following parts and experiments.


Figure 1 The sketch map of the characterization plasmid of nfGFP


To avoid misunderstanding, we show the diffenences between two nfGFP parts in our project. When measuring the degradation rate of nfGFP, we used the inducible promoter Pgal and expressed it on a plasmid. In contrast, when verifying the formation of CREATE with nfGFP, the promoter is TDH3 which expresses nfGFP frequently, and we integrated it in the chromosome by homologous recombination.

Experimental design


Experimental material: S. Cerevisiae with the characterization plasmid of nfGFP
Before inducing, the cells were cultured and enriched in the medium for 15 hours. Then we measured the OD600 values and the nfGFP fluorescence values. When the nfGFP fluorescence signals reached a steady value, we stopped inducing and changed cells to standard medium. Considering the end of induction as time zero, we recorded the changes of OD600 and nfGFP fluorescence signals every two hours. Due to the shutdown of nfGFP expression in the cell, cells can no longer produce reporter proteins, so the change in fluorescence signal intensity since then roughly reflects the degradation rate of nfGFP. By measuring the nfGFP fluorescence values, we plotted the degradation curves of nfGFP and calculated the half-life of nfGFP.

Experimental operation


(1) Enrichment and induction: Due to a large amount of culture medium required for subsequent measurements, we used shake flasks with 25 ml of induction medium to minimize the effect of the volume changes of the culture medium.
(2) Measurement of OD600: We used a UV spectrophotometry to measure OD600. To ensure data reliability, we unified the dilution multipliers while ensuring that the values are between 0.1-1.0 and as far as possible between 0.2-0.8.
(3) Measurement of fluorescence value: We use a Microplate Reader to measure the nfGFP fluorescence value. After the OD600 measurement, we wash off the culture medium and resuspend the cells with sterile water. Then, add 200 μl sample to each well of the 96 well microtiter plate. We set blank reference (water), control groups and experimental groups in three parallel groups when adding samples. The excitation/emission light wavelength of nfGFP in the Microplate Reader was 488/535 nm.

Data analysis and processing


Since the sample is diluted, the fluorescence intensity is equal to the measured fluorescence intensity multiplied by the dilution factor. Besides, the measured fluorescence intensity value should also subtract the blank value. In summary, the formula for calculating the nfGFP fluorescence intensity should be as follows.

Figure 3

 V': the true fluorescence intensity of nfGFP/200μl sample
 V: the measured value of nfGFP fluorescence signal/200μl sample
 C: blank reference(water)
 ω: the dilution factor of sample

The values of the three parallel groups of samples were averaged and plotted, the calculated data are shown in the figure.We use the following formula to fit the measured value and calculate the half-life value.


formula2

 N: instantaneous fluorescence value
 N0: initial fluorescence value
 τ: half life value


Figure 2 The degradation curve of nfGFP

Results analysis


From the results, it is observed that: When we stop inducing, the nfGFP signal decreased fast, indicating that nfGFP was rapidly degraded in the cells after the gene was no longer expressed.
The data shows that the half-life of nfGFP is around 7.558h, which is significantly reduced compared to the half-life of GFP, confirming the validity of our design.

Characterization of the formation of CREATE

Observe the disappearance of the nucleic acid dye

Experimental principle & purpose


In order to strictly control the expression of cas9 and further improve the chromosome cleavage efficiency, we designed version 2.0 of the cleavage element: 7flip + cre cleavage system (you can click link to the design page for details).
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.

Experimental design


Control group: Saccharomyces cerevisiae 4742 nfGFP
Experimental group: Saccharomyces cerevisiae 4742 nfGFP with 7flip+Cre plasmid ( cleavage system 2.0)
We placed the experimental and control groups’cells in the corresponding induction medium on the 96 well microtiter plate. Then, the cells were stained with Hoechest dye and placed under a fluorescent microscope lens for a fixed field of view for continuous filming to observe changes in intracellular DNA in the same field of view. According to the hypothesis, if it is indeed CREATE, the DNA will be progressively degraded, and its nuclear fluorescence signal will disappear, while the control group will remain unchanged. We will directly characterize the disappearance of DNA by the results of the dye staining shots.

Experimental operation


(1) Add samples: The cells were directly picked from the solid medium and added to the corresponding medium on the 96 well microtiter plate. We place 200μl sample of the medium in each well. We set blank reference (water), control groups and experimental groups in three parallel groups when adding samples.
(2) Staining: Cells were stained with Hoechst dye for 30 min according to the protocol, in the proportion of 1μl dye/200μl sample.
(3) Filming: To ensure a fixed field of view of the cells, after the suspended cells have settled and are motionless, they are placed under the fluorescence microscope lens for continuous fixed filming. According to the dye's protocol, the fluorescence microscope's channel settings can be the same as those of the commonly used nucleic acid dye DAPI. One photograph was taken every 30 min for 18 h. Since only one lens was available simultaneously, the experimental group was photographed continuously, while the control group was photographed only twice at the beginning and the end.

Results analysis


Hoechest is a nucleic acid dye that may bind to the RNA in the cell, resulting in a diffuse blue fluorescence inside the cell, while the very bright dots (indicated by arrows) in the cell indicate the location of the nucleus. This phenomenon can be identified in the pictures of the control group, which indicates that our dye is working correctly.
The following two figures show the changes in the control group (Saccharomyces cerevisiae 4742 nfGFP) after 18h of staining with Hoechst dye.


Figure 3 Control group stained by Hoechest 0h

Figure 4 Control group stained by Hoechest 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 Experimental Group stained by Hoechest 0-18h

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%.



The decline of the nfGFP fluorescence signal

Experimental principle & purpose


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 link.

See the BBa_K3939111

We expressed nfGFP with a frequently expressed promoter and integrated the part on the chromosome by homologous recombination. After that, we induced both experimental and control cells in culture. Since the genome of CREATE is disrupted by cleavage and no new nfGFP can be expressed, the fluorescence signal of CREATE will be lost soon with the rapid degradation of the original nfGFP. In contrast, normal cells in the control group can continuously express GFP, and the fluorescent signal intensity does not diminish. This difference can reflect whether the genome of the cells is degraded or not.


Figure 5 The sketch map of nfGFP integrated on chromosome

Experimental design


Control group: Saccharomyces cerevisiae 4742 nfGFP
Experimental group: Saccharomyces cerevisiae 4742 nfGFP with 7flip+Cre plasmid ( cleavage system 2.0)
We placed the experimental and control groups’cells in the corresponding induction medium on the 96 well microtiter plate and placed them under the fluorescent microscope lens with a fixed field of view for continuous photography to observe the changes in the nfGFP fluorescence signal produced by the cells in the same field of view. According to the hypothesis, DNA is gradually degraded during the generation of CREATE and cannot continuously express nfGFP; while the original nfGFP will be rapidly degraded. So the fluorescent signal will decline rapidly. In contrast, the control group can continuously express nfGFP with little change in signal intensity. We could confirm that by the results of fluorescence microscopy.

Experimental operation


(1) Add samples: The cells were directly picked from the solid medium and added to the corresponding medium on the 96 well microtiter plate. We place 200μl sample of the medium in each well. We set blank reference (water), control groups and experimental groups in three parallel groups when adding samples.
(2) Filming: To ensure a fixed field of view of the cells, after the suspended cells have settled and are motionless, they are placed under the fluorescence microscope lens for continuous fixed filming. The channel settings of the fluorescence microscope can be the same as GFP channel. Each photograph was taken every 30 min for 18h. Since only one lens was available simultaneously, the experimental group was photographed continuously, while the control group was photographed only twice at the beginning and the end.

Results and Analysis


The following two figures show the changes in the control group (Saccharomyces cerevisiae 4742 nfGFP) in the nfGFP channel after 18h of induction.


Figure 6 The fluorescence signal of Control group 0h



Figure 7 The fluorescence signal of Control group 18h


It can be observed that the number of cells increased, and the nfGFP intensity was almost as same as begining, which indicates that the continuous production and degradation of nfGFP in the control group’s cells reached a balance, the nfGFP part works properly, and the cell growth condition was proper.
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 video


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 degraded the chromosome, and thus cells can no longer express nfGFP, which indicates that our cleavage system works appropriately.
2.The half-life of nfGFP is short, so 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 credible. In the meantime, since the mechanism of the degradation of nfGFP involves organelles and enzymes, it also reflects that CREATE still has cellular mechanism, organelles and metabolic activity despite its genome was degraded.
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 8 Experimental group in GFP channel 18h



Figure 9 Experimental group in normal channel 18h


The results showing that although CREATE could no longer express nfGFP, the cell structure remained intact.
To summarize the analysis above, we believe that CREATE is indeed being formed. Most of the CREATEs have complete cellular structures and remain the cellular mechanisms and metabolic activity. This important conclusion predicts that CREATE has the primary conditions to perform cellular functions and is the premise for future applications.

Measurement of growth curve

experimental principle & purpose


We used the differences in the growth curves of the experimental (CREATE) and control (without the cleavage system) groups to show that our cleavage system worked successfully so that CREATE 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 spectrophotometry 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 groups of cells. 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.

Experimental designe


Control group:Saccharomyces cerevisiae
Experimental group 1:Saccharomyces cerevisiae with Delta plasmid(cleavage system 1.0)
Experimental group 2:Saccharomyces cerevisiae with 7flip plasmid +Cre plasmid(cleavage system 2.0)
We measured the OD600 values of the experimental and control groups after 15 hours of enrichment in the corresponding medium and diluted them to obtain the exact cell concentrations, which were then transferred to the corresponding induction media. Considering this as time zero, we measured the OD600 value of three groups of cells every two hours and plotted the growth curve.

Experimental operation


(1) Enrichment and induction: Due to a large amount of culture medium required for subsequent measurements, we used shake flasks with 25 ml of induction medium to minimize the effect of the volume changes of the culture medium.
(2) Measurement of OD600: We used a UV spectrophotometry to measure OD600. To ensure data reliability, we unified the dilution multipliers while ensuring that the values are between 0.1-1.0 and as far as possible between 0.2-0.8.

Results analysis


Figure 3

Figure 10 Raw data of OD600 of three groups of cells


Figure 3

Figure 11 The growth curve of three groups of cells


From the figure, cells in two experimental groups whose chromosomes were degraded didn’t significantly increase in yeast population compared to cells in control group. The results coincides with our hypothesis that after cells’ chromosomes were degraded, they cannot grow and reproduce any more, so the growth curve of the colony in both experimental groups should be below the control group’s.
The growth curve of cells with 7flip plasmid and Cre plasmid(cleavage system 2.0) is below the growth curve of cells with Delta plasmid (cleavage system 1.0), which shows that our improvement of the cleavage system is effective.

Flow cytometric analysis and sorting

Experimental principle & purpose


Flow cytometer is a device that enables rapid analysis and sorting of cells at single-cell level. It can measure, store and display a range of biophysically and biochemically critical characteristic parameters of dispersed cells suspended in liquid and sort specified cells from cell subpopulations based on a pre-selected range of parameters.
The following section will describe how we set parameters during analyzing and sorting of CREATE with flow cytometry. We set limits on a range of parameters such as cell morphology, dye staining signal, percentage of cell populations, and finally determine the threshold for CREATE from the cell population.
a) Cell morphological parameter: Depending on the cellular morphology, yeast cells with normal cellular morphology can be framed and distinguished from cell debris.


Figure 12 The setting of Parameter 1(P1) of Flow cytometer


b) Cell morphological parameters FSC-H and FSC-A: Based on the previous step, we selected cells near the diagonal in the image by making a plot based on the cell morphological parameters FSC-H and FSC-A. In this way, single cells can be screened out from the cell population.

Figure 13 The setting of Parameter 2(P2) of Flow cytometer


c) PI dye signal: PI is a membrane-impermeable nucleic acid dye that cannot enter cells with intact cell membranes, which can be used to determine whether a cell is alive or not. Based on the previous step, the cell morphology parameter SSC-H and PI-A intensity in response to the PI dye can be used to distinguish live cells from dead cells.

Figure 14 The setting of Parameter 3(P3) of Flow cytometer

The cells in the top right of the image are the result of this step.


d) DRAQ5 dye signal: DRAQ5 is a membrane-permeable nucleic acid dye that can be used to determine whether a cell has chromosomes. Based on the previous step, cells with low intensity of response to DRAQ5 (0.0025 of normal cells) were selected.
According to the above signals and parameters in control (without cleavage system) cells, we determined the threshold where CREATE cells are located, referred to as the "CREATE Gate".

Figure 15 The setting of Parameter 4(P4) of Flow cytometer

According to the above signals and parameters in cells in control groups(without cleavage system), we determined the threshold where CREATE cells are located, referred to as the "CREATE Gate".

Experimental designe


Control group:Saccharomyces cerevisiae 4742 nfGFP
Experimental group:Saccharomyces cerevisiae 4742 nfGFP with 7flip+Cre plasmid (cleavage system 2.0)
We enriched cells in experimental and control groups in the corresponding medium for 12 hours and then transferred them to the induction medium. Before starting the flow cytometric analysis, the samples are stained. Moreover, the "gate" of CREATE is determined by the staining reaction's fluorescence signal and morphological parameters of cells in Control group. This "gate" is used to analyze and sort the cells in the experimental group.

Experimental operation


(1) Nucleic acid dye staining: The samples were stained with PI and DRAQ5 for 30 min. 1.5 μl of diluted PI dye and 2 μl of diluted DRAQ5 dye were added to each 200 μl sample.
(2) Parameter setting: After the microfluidic flow rate of the flow cytometer has stabilized, the parameters are automatically set and "gated" by the computer's reserved parameters.


Figure 16 Our advisor showed us how to use flow cytometer

Results analysis


The following two figures show the results of the flow cytometric analysis.


Figure 17 The formation rate of cells in Control goup by Flow cytometer analysis/Figure 18 The formation rate of cells in Experimental goup by Flow cytometer analysis


The left graph is the control group, and the right is the experimental group. According to the results, our highest formation rate of CREATE reached more than 80%.

During the iteration of the project, we considered using the Saccharomyces cerevisiae SY14 strain, which has only one chromosome, as a chassis to make CREATE. We conjectured that SY14 might form CREATE more efficiently. We used flow cytometric analysis to compare the effeciency 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 19 The formation rate of CREATE in different stains by Flow cytometer analysis


Point dilution plate and CFU dilution

experimental purpose


We used the differences of growth status in solid medium of cells in experimental (CREATE) and control (without the cleavage system) groups to show that our cleavage system worked successfully so that CREATEs was completely different from normal cells. If it was indeed CREATE, it could not grow and reproduce, while the normal cells could. We confirmed this difference and thus corroborated the formation of CREATE.
We also want to know whether the formation rate of CREATE would increases with more time of inducing.

Experimental designe


Control group1:Saccharomyces cerevisiae 4741
Control group2:Saccharomyces cerevisiae 4742
Experimental group 1:Saccharomyces cerevisiae with Delta plasmid(cleavage system 1.0)
Experimental group 2:Saccharomyces cerevisiae with 7flip plasmid +Cre plasmid(cleavage system 2.0)
We added inducer to the shake flask of the experimental group to induce Cas9 protein express and degrade chromosomes to generate CREATE, and no inducer was added to the shake flask of the control group. Culture them together under the same condition. Take samples from the experimental groups and the control groups every 12 hours to measure the OD600, and dilute with double distilled water at the same multiple.
After dilution, apply the same amount of liquid to the solid medium and take the same amount of bacterial solution (2ul) on the same piece of solid medium.

Experimental operation


(1) Measurement of OD600: We used a UV spectrophotometry to measure OD600. To ensure data reliability, we unified the dilution multipliers while ensuring that the values are between 0.1-1.0 and as far as possible between 0.2-0.8.
(2) Dilute: After measure the OD600, we draw 100ul bacterial liquid from cuvette to a sterile EP tube containing 900ul sterile double distilled water and dilute 10 times, then use the same method to dilute it gradually until the dilution factor is about 10^(-5), we dilute cells in four groups into the same concentration according to the OD600.
(3) CFU: We take 100ul of the bacterial solution on the solid medium and daub it equably.
(4) Point dilution plate: We take 2μl of the bacterial solution point on the solid medium. We put cells in the experimental groups and the control groups with same dilution ratio on the same line.
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.
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.

Results analysis


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 20 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 21 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


Shape

Experimental purpose


To investigate whether cell morphology is affected when chromosomes are removed.

Experimental design


Control group:Saccharomyces cerevisiae 4742 nfGFP
Experimental group:Saccharomyces cerevisiae 4742 nfGFP with 7flip+Cre plasmid (cleavage system 2.0)
After 15 hours of enrichment in the corresponding medium, we transferred them to the corresponding induction medium. After another 18h, we observe the cell morphology of cells in control and experimental groups under a light microscope.

Result and analysis




Figure 22 Cells in control group observed by Microscopy



Figure 23 Cells in Experimental group observed by Microscopy


Compared to cells in the control group, 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 change within a short period.
Another result that can support this conclusion is that in experiments 3.2 when removing the GFP channel, we can see that cells without nfGFP fluorescence in vivo (CREATE) remain intact morphological structure.

References

[1]John R. Houser,Eintou Ford,Sudeshna M. Chatterjea. (2012). An improved short‐lived fluorescent protein transcriptional reporter for Saccharomyces cerevisiae. Yeast(12):519-530.
[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.

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About Us

School of Chemical Engineering and Technology, Tianjin University, 135 Yaguan Road, Jinnan District, Tianjin