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Team:UT-Dallas/Modeling
From 2014.igem.org
MODELING
We utilized the CRISPR/Cas system with gRNA engineered to recognize genes from infectious bacteria using bacterial specific phages delivery system. Specifically, we target Cholera as a proof of principle. To target the Choleras genome, our team focus on (1) cutting efficiency of the gRNA-CRISPR/Cas9 system and (2) the delivering efficiency of the phage delivery system.
There are two important features we have to look at:
- 1. On the Intracellular level, in a single Cholera cell, the CRISPR/Cas9-gRNA system is transcript and translated. The complex then cleaves the Cholera native genome, leading to cell death.
- 2. On the population level, the phagemid deliver the CRISPR/Cas9-gRNA plasmid from the probiotics to
CELLULAR LEVEL
The model focus on simulating our experiment, with the decrease YFP signal observed as a signal of cell death.
Each of the components in our system is transcript with a separate promoter. Our further goal with the model is to determine the more efficient promoter by varying transcription rate.
Design - This part describes the design of the model.
We focus on the concentration of total count of free molecules transcript and translated in a cell versus time.
- (1) Active Cas9 system induced by the presence of Dox molecule
- (2) Inactive Cas9 system induced by the absence of Dox molecule (inhibited by TetR repressor protein)
Figure 1. Intracellular Model with large amount of Dox and active CRISPR system from minute zero-th to 1400th
(a) Cas9 is continuously being transcript, thus a logarithmic growth of mRNA molecules of Cas9 (blue line, mRNA-Cas9) is observed. Counterbalanced by natural degradation rate, the total count of mRNA-Cas9 reaches a stable population and switches to a plateau.
Cas9 Protein (green line, pro-cas9) total count is keep at zero, due to immediate conversion into CRISPR complex.
(b) gRNA (blue line) total count grows logarithmically and reaches a plateau due to natural degradation. The size of gRNA is 7 times smaller than Cas9, thus it is transcript much faster than Cas9. Thus the CRISPR complex (green line, Cas9-gRNA) is much lower in comparison with the blue line.
(c) This figure shows the huge starting amount of Dox (red line, Dox) for this simulation 10e11. Dox is not regenerated and only goes down due to natural degradation and binding with TetR protein (green line, pr-tetR) to form Dox-TetR Complex (cyan line, Dox-tetR). All the other amount are too low in relation to Dox, thus harder to observe in the figure. (Go here for a better look at mRNA of TetR, TetR protein, and Dox-TetR Complex: https://static.igem.org/mediawiki/2014/0/03/UTDallas_Figure1SCALE_DOWN.jpg)
(d) Yellow Fluorescence Protein (green line, pr-YFP) go through a logarithmic growth, reaching a peak, and then degrades. Total count molecules mRNA of Yellow Fluorescence Protein (blue line, mRNA-YFP) observes a much higher logarithmic growth and then degrades really fast as the transcription is stopped by the YFP-DNA cleaving of CRISPR Complex.
CAS9 INACTIVE
Figure 2. Intracellular Model without Dox and Inactive CRISPR system from minute zero-th to 1400th
(a) Cas9 is transcript with a rapid logarithmic growth of total free mRNA molecules of Cas9 (blue line, mRNA-Cas9) then quickly declines into logarithmic death phase. Cas9 Protein (green line, pro-cas9) total count is keep at zero as no mRNA is available for translation.
(b) gRNA (blue line) total count grows logarithmically and reaches a plateau due to natural degradation. The size of gRNA is 7 times smaller than Cas9, thus it is transcript much faster than Cas9. Thus the CRISPR complex (green line, Cas9-gRNA) is much lower in comparison with the blue line. No formation of Cas9 Protein implies no binding of Cas9 and gRNA, implies no formation of CRISPR complex.
(c) No Dox (red line, Dox) is added in this simulation. Thus TetR protein (green line, pr-tetR) is not binded with dox to form Dox-TetR Complex (cyan line, Dox-tetR), therefore Dox-TetR Complex is always zero. TetR protein and mRNA of TetR total count grow logarithmically and reach the plateau phase due to natural degradation.
(d) Without the effect of CRISPR Complex, Yellow Fluorescence Protein (green line, pr-YFP) and molecules mRNA of Yellow Fluorescence Protein (blue line, mRNA-YFP) grow logarithmically and reach the plateau phase due to natural degradation.
MODEL 3
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TRIVIA
All of the Mathematical Modeling works was done by Tra. She did not have any experience on programming prior to iGEM. Such dedication and diligence! It was thanks to the help and instructions from Dr. Ma and Taek that she was able to complete both the simulation and the model. Tra’s biggest lesson learnt from modeling experience, besides from knowing how to build mathematical models and programming, is to always back up her files in several places. Tra saves everything, all her works (and photos), in a flash drive. She always carries it with her, so there is no way she could lose the drive and its files. Two nights before the Wiki Freeze deadline, she was working late at our lab building. It was a beautiful night. The cold front brought nice chill to the Texas heat. The starless night sky was brightly lit up with the lights from the construction site next to our lab. It was midnight. Tra quickly packed up her bag and bike home. She got home, opened her bag, and found her USB broken in half. The electrodes were teared off. Her USB was crushed inside her bag. Let’s not discussed how that happened. But she tried a great deal with her EE friend to save the USB and savages the data inside. Sadly, at 4AM, the day before the deadline, they have to say goodbye to the USB. It was a great lost. The models and simulation you saw in this wiki was rebuilt from the vestiges. Cheers!
