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.
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
Figure 11 The growth curve of three groups of cells
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.
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