Zachary Miller BME 100 Computer Project Answers 1. units on various constants a.

constant: k nM/s Derivation: consider where r has units of nM/s, if the upstream repressor

b.

c.

d. e. f. g.

concentration is assumed to be 0 then rate is equal to k meaning k must have units of nM/s. constant: k0 nM/s Derivation: k0 is standalone meaning its units must match the units of the output nM/s constant: nM Derivation: given has units of concentration, k has units of nM/s, and the net units of the hill equation must be nM/s meaning must have units of concentration to cancel out the other concentration components leaving units of nM/s for the overall hill function. Constant: Derivation: first order reaction Constant: n unit-less Constant: Derivation: first order reaction Constant: Derivation: first order reaction

2. Non-Dimensionalization Given , and = + and t to yield: = This yields the

1. Find the non-dimensionalized derivative by taking the derivative of and 2. Given , rearrange to give (

, substitute this expression into the Hill function in each , cancel all leaving

following expression:

, and after some algebra:

3. Given

, rearrange and substitute this into the last term:

, this leaves

4. Putting all the terms together gives: ( = + + finally yields

and multiplying this expression through by = + +

Thus we conclude that : * = 5000 (note the exponent:-1) = =5

Note both expressions are unitless as required. 3. values of dimensionless (unitless) constants: =5

4. Matlab Code

function repressor clear all [T,Y] = ode45(@repressilator, [0:60:150*60], [5 1 5 2 5 3]); %ODE solver uses Runga Kutta to solve %non-linear ODEs under function repressilator, note sampled every nondimensionless minute %instead of non-dimensionless second because otherwise graph sheet of blue %for oscillatory case subplot(2,3,1); semilogy(T, Y(:,1)); xlabel('Time'); ylabel('mRNA TetR'); title('mRNA Tet R vs. Time') subplot(2,3,2); semilogy(T, Y(:,2)); xlabel('Time'); ylabel('Protein TetR'); title('Protein TetR vs. Time') subplot(2,3,3); semilogy(T, Y(:,3)); xlabel(' Time'); ylabel('mRNA Lambda Cl'); title('mRNA Lambda Cl vs. Time') subplot(2,3,4); semilogy(T, Y(:,4)); xlabel(' Time'); ylabel('Protein Lambda Cl'); title('Protein Lambda Cl vs.Time')

subplot(2,3,5); semilogy(T, Y(:,5)); xlabel('Time'); ylabel('mRNA LacI); title('mRNA LacI vs. Time') subplot(2,3,6); semilogy(T, Y(:,6)); xlabel('Time'); ylabel('Protein LacI-cl'); title('Protein LacI vs. Time') function dy=repressilator(t,y) % sustained oscillations as suggested by Elowitz paper: n = 2, alpha0/alpha = 103. a=5000; %ratio of 10-3 a0=5; n=2; %hill coefficient B=1; % steady state, use values given in lab handout %a=5000; %ratio of 10-3 %a0=5; %n=2; %hill coefficient %B=.2 %y(1)=tetr-protein, y(2)=tetr-mRNA, y(3)=lambda-cl protein, y(4)=lambda-cl %mRNA, y(5)=lacI-protein, y(6)=lacI-mRNA dy=zeros(6,1); dy(1) = (a./(1+(y(6)).^n)) + a0 - y(1); %tetr repressed by lacI transcription y(6) dy(2) = B.*(y(1) - y(2)); dy(3) = (a./(1+(y(2)).^n)) + a0 - y(3); %lamda-cl repressed by tetr hence y(2) dy(4) = B.*(y(3) - y(4)); % dy(5) = (a./(1+(y(4)).^n)) + a0 - y(5); %lacI repressed by lamda-cl y(4) dy(6) = B.*(y(5) - y(6)); end

end
5. Sustained Oscillations

Figure 1: Sustained Oscillations of the repressilator system were initiated using alpha=5000, alpha0=5, n=2, B=1 in accordance with the Elowitz paper Figure 1B phase space plot. In order to initiate an unstable equilibrium, it was required that alpha, the level of allowable protein production, divided by alpha0, the leakiness of the promoter, was 1e-3. B is bounded for sustained oscillations for small n, but as n increases, B can take on any value and sustained oscillation will still occur. Further analysis found that B=10 with all other parameters left the same leads to a damped oscillation, but as n is increased, the damped scenario becomes sustained. The oscillation periods are actually much shorter than shown in the graph above, but the graph was sampled every 60 nondimensionalized seconds to make the graph cleaner because otherwise it was a solid blue without clear limit cycles as shown below (figure 2). In figure 1A: tetR mRNA is

inhibited by lacI protein, figure 1B simply represents the conc. of net tetR protein produced. Figure 1C describes lambda-cl mRNA inhibited by tetR protein. Figure 1e describes lacI mRNA inhibited by lambda-cl protein.

Figure 2

6. Steady State

Figure 3: Steady State was attained using values given in lab handout where alpha=5000, alpha=5, n=2, beta=.2, thus all parameters were left the same except for beta which was decreased. Note that as beta is decreased sustained oscillations remain until reaching the beta=.2 threshold where the unsteady limit cycle region merges into the steady state region of the phase space. In figure 3A: tetR mRNA at conc. of P(1)=1 is inhibited by lacI protein. Figure 3B simply represents the conc. of net tetR protein produced. Figure 3C describes lambda-cl mRNA at conc. of P(2)=2 inhibited by tetR protein. Figure 3e describes lacI mRNA at conc. of P(3)=3 inhibited by lambda-cl protein.
. Further Comments on Model The non-linear differential equation solutions using phase space analysis offer effectively three different solution types depending on parameter values: steady state, damped oscillation to steady state, and sustained oscillation. Sustained oscillations in repressilator can be understood by examining figure 1. TetR is inhibited by its upstream repressor Lambda Cl. In figure 1, it is clear that as lambda cl protein concentration decreases

slightly due to repression of lambda-cl production by lacI that TetR production increases. But because LacI is inhibited by increasing amounts of TetR protein, it allows for increasing levels of lambda Cl to feedback and inhibit TetR. At the correct concentration values, this causes sustained oscillation because the amount of repression vs. the amount of activation is unbalanced. Stable Steady State occurs when repressor binding and activation are exactly balanced leading to fixed equilibrium protein/mRNA concentrations over long times.