Part:BBa_K3939666
Gal Cas9
The Cas9 proteins are regulated by Gal. Tianjin 2021 uses the Cas9 proteins to cut the chromosomes of eukaryotic cells. The highlight is that Gal regulates the expression of the Cas9 protein, which allows us to control the time of cutting yeast chromosomes.This part is our first version of cutting system.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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 708
Illegal BglII site found at 1645
Illegal BamHI site found at 2463
Illegal XhoI site found at 4001 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 3197
Illegal AgeI site found at 85 - 1000COMPATIBLE WITH RFC[1000]
Measurement of growth curve
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）
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
From the figure, cells in experimental group 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 experimental group should be below the control group’s.
Point dilution plate and CFU dilution
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 4741
Experimental group1: Saccharomyces cerevisiae 4741 with Delta plasmid(cleavage system 1.0)
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.
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.
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.
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