Team:UT-Dallas/Modeling

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

This simulation provides a simple and intuitive visualization of the effects of the probiotics onto the pathogenic Choleras population.
Initially, we plan to test on Phagemid M13 and then CTXphi, which is more specific to Cholerae. (Please refer to our experiment design page). Our lab had ordered Cholerae for this experiment. However, the limited time did not allow us to test this. After Wiki Freeze, we still expect to continue the experiment.
Nevertheless, this is where this population simulation comes in and shows the potential application of our bio-system.

POPULATION LEVEL DESIGN

This part describes the design of the simulation, how we come up with it and its relation to our wet lab experiment.
Our design starts with a relatively stable probiotics population (green) and a growing pathogenic Cholerae population (black). The probiotics should be able to sense the presence of Cholerae with a sensing mechanism. (We discussed with Colombia iGEM Team on collaboration on this subject, as they were working on the sensing specificity for Cholerae. We invite you to visit their wiki and check out their project.)
Ultimately, we design the probiotics with our genetically modified plasmid with gRNA-Cas9 system to be a stable non-pathogenic colony growing in harmony with the human intestinal flora.

• [mode 0] In the absence of Cholerae, the probiotics would not release phages.
• [mode 1] In the presence of Cholerae, the probiotics would release phages with the CRISPR system plasmid.

The molecules released by Cholerae induces the production of phages (the build up of phage capsid protein), the packaging of our CRISPR system plasmid, and the release of such phages.Once the phages get in contact with Cholerae cells, the plasmid is released into the Cholerae cytosol. Thus, the cleaving system goes to work and the bad Cholerae die! (To know more on our system mechanism, please visit our project mechanism page.) Once the Cholerae and its presence-sensing-signal died out, the probiotics return back to [mode 0] and stop releasing phages.

POPULATION LEVEL SIMULATION

We use GRO - The cell programming language, a shareware developed by The Klavins Lab at the University of Washington.

• Green population := the probiotics (at time 0, population = 36)
• Black population := the pathogens (V. Cholerae) (throughout the simulation, population = 36)
• Blue signal := signal emitted by the pathogens, indicating its presence (at time 0, population = 15)
• Pink signal := the phages emitted by the probiotics, carrying the CRISPR plasmid

POPULATION LEVEL RESULT

The delay in signal disappearance indicates the significance of choosing appropriate phages for the delivery system with appropriate speed, effectiveness, specificity, and degradation rate.