Difference between revisions of "Template:Team:Utrecht/MainBody"

The first endonuclease activity assay was used to determine the optimal concentration of gRNA for the assay with the secreted proteins. The concentrations used were all functional for the Cpf1 protein (Figure 5). Cas9 however, showed no cleavage of the targeted DNA at all. This may be due to low quality gRNA. In the second assay, which contained sCas9 and sCpf1 purified from medium, there was no cleavage visible at all, even for the controls with Cas9 and Cpf1, as shown in figure 6.  

<center><img style="margin-top: 10px;" src="https://static.igem.org/mediawiki/2017/f/fc/UU_secretion_fig5.png"></center>

Wherein S’ is the increase in signal, given by the probability of cleavage (pc) for the remaining uncleaved complex. The remaining uncleaved complex is given by the remaining complex fraction (C) and how likely it is that it has not already been cleaved (one minus the integral of pc from 0 until that timepoint).

When we solve this for each Protease Chain version and for both the transient and substrate-mediated complex, we end up with time-plots of the signal contribution of a single binding event. In these plots, we see that the resulting substrate-mediated complex signal for the slow cleaver (top) is about half as strong as the signal for the fast cleaver (bottom).In theory, this is not a problem since the signal can be amplified by the cells.  

Using the contributions of a single transient binding event and a single substrate-mediated binding event, we can calculate a proxy for precision by dividing the contribution of the true signal (substrate-mediated) by the contribution of the false signal (transient). For the slow rate, the contribution of a single substrate-mediated event is almost 48 000 times that of the transient occurrence. For the fast cleavage rate, this is only 17 times.

There are eight important parameters in these equations. The k1 and k2 rates are the association and dissociation rates between DNA and the target chain. The k3 and k4 are the association and dissociation rates of the protease chain and DNA. k5 gives the effective cleavage rate. pP and pT are the production rates of the protease and target chain and d gives the decay-rate for proteins in general.

The concentration of each state is given by the name of the state in square brackets, such that T stands for target chain, S stands for substrate (DNA), P stands for protease chain, F stands for effector molecule and Tc stands for cleaved target chain. Binding is indicated by ‘:’ between two components.  

The first thing we did was to check whether this system of equations behaves the way we expect it to, on short timescales. At first, we were only interested in the system equilibria. At the time-scale of state transitions, protein production and decay per time-unit are negligible and so the values for d, pT and pP were initially set to zero.

Using the association and dissociation constants from Richardson et al. <i class="ref" data-id="3">3</i>, we set the parameters k3 and k4 to 4 * 104 and 5 * 10-5 respectively. Fonfara et al. <i class="ref" data-id="4">4</i> found a range of Cpf1 affinities in the same order of magnitude as Richardson et al. and so we chose to perform several runs with a variety of Cpf1 parameters going from .9 to 1.1 times the rates of Cas9. All runs resulted in similar equilibria where all target chain would be processed and cleaved.