Team:Nanjing High School/Model
Model
Genetic circuits
Here, we develop a type of micro-organism that can record the length of time it is exposed to blue light. We find that Cas1-Cas2 complex which proteins in the process of cutting and inserting DNA fragments into CRISPR array, can be restrained by Thermobifida fusca (Tfus) Cas3. We construct plasmids with Cas1-Cas2 and Cas3 expression, and the Cas3 is built downstream of FixK2 promoter controlled by blue light sensor. Therefore, a quantitative relationship between the acquisition of new spacers of the CRISPR array in Cas1-Cas2 and the length of time that the bacteria are exposed in blue light is developed. Combining this technique with practical applications will yield a method to measure time of blue light emitting.
However, for simplicity,we refer to other teams’ model (UNITN-Trento) and want to see if it is fit our experiement.
Some kinetic constants have been found in literature and used as center of an interval to estimate the variables of our model, other variables were estimated from hypothetical data within constraints of our choice:
Starting DNA concentrations were estimated based on hypothetical data within the interval [1e-6;1e-2]
FixJ phosphorylation and FixJ-P de-phosphorylation kinetics, k4 and k4b, were estimated based on hypothetical data within the interval [0;10] 1/s
Transcription kinetic was taken from BIONUMBERS (k = 0.33 1/s) and estimated for each reaction inside the interval [1e-4;1e-1] 1/s
RNA degradation kinetic was taken from BIONUMBERS (k = 0.011 1/s) and estimated for each reaction inside the interval [1e-3;1e-1] 1/s
Protein degradation kinetic was taken from BIONUMBERS (k = 0.00083 1/s) and estimated for each reaction inside the interval [1e-3;1e-1] 1/s
Protein translation kinetic was taken from BIONUMBERS (k = 22 1/s) and estimated within the interval [1e-2;1] 1/s
The autophosphorylation kinetics in the light and in the dark, k3 and k3b, were taken from (Möglich A, J MOL BIOL, 2009, 385(5):1433-44)
The result of a time-course simulation of 2e+05 seconds with a 8e+04 dark exposure window is shown in Figure 2.
Figure 2: the image shows the protein concentration over time. Cas3 is produced only during the window of dark exposure.
Calculate the percentage of DNA inserts
Image J
1 File →open →TIF
2 Image →Type→ 8-bit
3 Edit →Invert
4 Process→Subtract Background. In the pop-up dialog box, fill in the 50 basics, and tick the Light background, click OK.
5 Analyze→Set Measurements. In the pop-up dialog box, select Area, Mean gray value, Min & max gray value, Integrated density.
6 Analyze→Set Scale. Fill in the pop-up dialog box "Unit of length" followed by "pixels", the other without modification.
7 Edit → Invert
8 Select a series of irregular square areas under the menu bar and choose one depending on the situation. Then pull the square down the first strip and circle the strip as much as possible.
9. Click on the measurement that appears in the menu bar Analyze to pop up the grayscale statistics for your selected area.
10. Manually move the rectangle to the next strip and repeat steps 8 and 9 until all strips have been measured.
11. When all the strips have been measured, select “Select All” of “Edit” in the result and select File → Save as to export the excel form directly.