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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
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
Figure 3. Intracellular Model with a small amount of Dox and Inactive CRISPR system from minute zero-th to 1400th
We are mainly interested in figure 3.(c) which shows the interaction between mRNA of TetR, Protein TetR, binding Dox molecules, and Dox-TetR complex.
Refer to Figure 1 for Figure 3.(a), 3.(b), and 3.(d) interpretation.
(c) Dox (red line, Dox) goes down from a moderate amount of 900 to zero at time step ~320. TetR protein (green line, pr-tetR) amount from time step 0 to ~320 is zero due to binding with dox to form Dox-TetR Complex (cyan line, Dox-tetR), which increases.
From time step ~321 to 1400, Dox is zero, therefore Dox-TetR Complex changes into degradation phase as it is no longer being produced (no Dox to bind with TetR). Free TetR proteins start to increase from time step ~321. Without effect on TetR transcription, mRNA of TetR (blue line, mRNA-tetR) total count grow logarithmically and reach the plateau phase due to natural degradation.
POPULATION MODEL
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