Difference between revisions of "Part:BBa K3939998"

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Cas9 with flipped promoter

Brief introduction

This part is constructed by two LoxP sequences and a reversed pGal promoter. the pGal promoter is not a tightly regulated promoter. thus,we used estrogen and galactose to induce Cas9 expression, allowing this element to be regulated more precisely. In our project, we use it to cut chromosomes to make chromosome-free eukaryotic cell-- CREATE. More details about this part, please click (Measurement). Sequence and Features

Characterization

Use fluorescent microscope to test the function of part

1.Experimental principle & purpose：
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.
2. Experimental design：
Control group: Saccharomyces cerevisiae 4742 nfGFP
Experimental group: Saccharomyces cerevisiae 4742 nfGFP with 7flip plasmid+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.
3. 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. 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.
4.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 18h, 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.
File:T--Tianjin--M6.mp4
In the video, it is observed that most of the fluorescence signal disappears, visualizing the process of degradation of the DNA, which indicates that the cleavage system works correctly in most cells but the efficiency of CREATE formation is not yet 100%.

The decline of the nfGFP fluorescence signal

1. 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 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

2. Experimental design：
Control group: Saccharomyces cerevisiae 4742 nfGFP Experimental group: Saccharomyces cerevisiae 4742 nfGFP with 7flip plasmid+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.
3. 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.
4. 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.

Video2 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 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.

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

1. 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.

2. Experimental design
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.
3. 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.

4. Results analysis

Figure 10 Raw data of OD600 of three groups of cells

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.

1. Experimental principle & purpose：
Flow cytometer is a device that enables rapid analysis and sorting of cells at the 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<i/> 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.
<i>Figure 14 The setting of Parameter 3(P3) of Flow cytometer 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.
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".
2. Experimental design：
Control group：Saccharomyces cerevisiae 4742 nfGFP
Experimental group：Saccharomyces cerevisiae 4742 nfGFP with 7flip plasmid+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.
3. 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.
4. Results analysis：
The following two figures show the results of the flow cytometric analysis.

Figure 16 The formation rate of cells in Control goup by Flow cytometer analysis Figure 17 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 18 The formation rate of CREATE in different stains by Flow cytometer analysis

1. 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.
2. Experimental design：
Control group1：Saccharomyces cerevisiae 4742
Experimental group1: Saccharomyces cerevisiae 4742 with Delta plasmid(cleavage system 1.0)
Experimental group2: 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.
3. 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.
4. Results analysis：
We can observe the different growth status between experimental groups and the control groups based on the results of the point dilution plate. This indicates that the part is in working and indirectly support the formation of the chromosomal-free eukaryotic cell CREATE.

Figure 19 Different growth status between the experimental groups and the control groups

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 20 Different colony counts between the experimental group and the control group
The left is the experimental group (delta G) 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.