The CI-Cro bistable switch, controlled by PRM and PR, present in the λ bacteriophage, controls the switching between the lytic and lysogenic states of the virus. In the lysogenic state, the CI repressor is produced in abundance, repressing PR from producing Cro, which is required to switch from lysogenic to lytic state. In the lytic state, the production of CI from PR is repressed by Cro (Schubert et al., 2007).
Here, we study the RNA production dynamics of the CI–Cro genetic switch as a means to indirectly validate that the observed changes with temperature in the dynamics of the genetic Repressilator are due to the temperature-dependence of the functionality of cI, a repressor of the promoter PR.
Previous studies suggest that the activity of CI is temperature dependent (Jana et al., 1999). In the switch system used here, the promoter PRM, which controls the expression of CI, also controls the transcription of a 96 MS2d binding array. The other promoter of the switch, PR, controls the expression of Cro. The activity of this promoter is not followed. From previous studies, it was observed that Cro–DNA interactions do not vary
were found to be similar (mean and standard deviation of around 1400 and 1100 s, respectively) when both are induced with 1% Arabinose, which is expected since the same intake system is used. Importantly, lac/ara-1, when induced with IPTG, exhibited a smaller standard deviation (700s), although it had a similar mean (approximately 1400s), which suggests that the intake process of arabinose is noisier than the intake process of IPTG. We also found that the mean tdiff is significantly affected by the concentration of arabinose in the medium (Mäkelä et al., 2013). Using a stochastic model of the intake process that accounts for the inducer concentration and the empirical distribution of t0, and that assumes that transcription initiation is a 3-stepped process, we further found that the intake step likely adds variability to the RNA numbers in individual cells during the transient stage to reach the equilibrium (i.e. maximum activation possible given the inducer concentration in the medium).
4.4 Dynamics of the CI-Cro switch
The CI-Cro bistable switch, controlled by PRM and PR, present in the λ bacteriophage, controls the switching between the lytic and lysogenic states of the virus. In the lysogenic state, the CI repressor is produced in abundance, repressing PR from producing Cro, which is required to switch from lysogenic to lytic state. In the lytic state, the production of CI from PR is repressed by Cro (Schubert et al., 2007).
Here, we study the RNA production dynamics of the CI–Cro genetic switch as a means to indirectly validate that the observed changes with temperature in the dynamics of the genetic Repressilator are due to the temperature-dependence of the functionality of cI, a repressor of the promoter PR.
Previous studies suggest that the activity of CI is temperature dependent (Jana et al., 1999). In the switch system used here, the promoter PRM, which controls the expression of CI, also controls the transcription of a 96 MS2d binding array. The other promoter of the switch, PR, controls the expression of Cro. The activity of this promoter is not followed. From previous studies, it was observed that Cro–DNA interactions do not vary
significantly between temperatures from 24–37°C (Takeda et al., 1992), thus, any behavioural changes observed in the switch with changing temperature in this range should arise from the changes in CI–DNA interactions. For this, time lapse microscopy was performed to obtain the RNA production intervals from the promoter for 2 hours, with images taken every minute. The measurements were conducted at 24 °C, 27 °C, 30 °C, 33 °C and 37 °C.
Table 2 shows the number of samples (i.e. intervals), mean and standard deviation of the intervals’ duration in each condition. It is observed that as temperature increases, the kinetics of production of the target RNA shifts from sub-poissonian (CV2 < 1) to super- poissonian (CV2 > 1). From Table 2 it is also observed that as the temperature increases, the mean interval between consecutive transcription events decreases. The significance of this change was verified by comparing the distributions of intervals in consecutive temperatures with the K–S test. Table 3 shows that the distributions at 24 °C and 27 °C cannot be distinguished from one another. A similar trend was observed in case of the distributions at 33 °C and 37 °C. Meanwhile, the distributions obtained from 27 °C and 30 °C, as well as distributions from 30 °C and 33 °C differs significantly from one another. This suggests that there is a change in the dynamics of transcript production around 30 °C, which is similar to the change observed in the behaviour of the Repressilator (Results in the next chapter).
T (oC) No. of intervals µ (s) σ (s) CV2 24 157 1242 1166 0.88 27 229 1152 1191 0.67 30 88 1130 1040 0.85 33 539 788 807 1.05 37 324 714 785 1.21
Table 2: Intervals between appearances of new RNA molecules in individual cells. Table shows
per condition, number of intervals, µ (s) is mean, σ (s) is standard deviation and CV2 is coefficient of variation
significantly between temperatures from 24–37°C (Takeda et al., 1992), thus, any behavioural changes observed in the switch with changing temperature in this range should arise from the changes in CI–DNA interactions. For this, time lapse microscopy was performed to obtain the RNA production intervals from the promoter for 2 hours, with images taken every minute. The measurements were conducted at 24 °C, 27 °C, 30 °C, 33 °C and 37 °C.
Table 2 shows the number of samples (i.e. intervals), mean and standard deviation of the intervals’ duration in each condition. It is observed that as temperature increases, the kinetics of production of the target RNA shifts from sub-poissonian (CV2 < 1) to super- poissonian (CV2 > 1). From Table 2 it is also observed that as the temperature increases, the mean interval between consecutive transcription events decreases. The significance of this change was verified by comparing the distributions of intervals in consecutive temperatures with the K–S test. Table 3 shows that the distributions at 24 °C and 27 °C cannot be distinguished from one another. A similar trend was observed in case of the distributions at 33 °C and 37 °C. Meanwhile, the distributions obtained from 27 °C and 30 °C, as well as distributions from 30 °C and 33 °C differs significantly from one another. This suggests that there is a change in the dynamics of transcript production around 30 °C, which is similar to the change observed in the behaviour of the Repressilator (Results in the next chapter).
T (oC) No. of intervals µ (s) σ (s) CV2 24 157 1242 1166 0.88 27 229 1152 1191 0.67 30 88 1130 1040 0.85 33 539 788 807 1.05 37 324 714 785 1.21
Table 2: Intervals between appearances of new RNA molecules in individual cells. Table shows
per condition, number of intervals, µ (s) is mean, σ (s) is standard deviation and CV2 is coefficient of variation
T (oC) 24 27 30 33
27 0.149
30 0.06
33 0.009
37 0.478
Table 3: P-values of the Kolmogorov-Smirnov test between distributions of intervals between
consecutive RNA production events from PRM. For p-values <0.01, the hypothesis that the distributions are the same is rejected.
T (oC) 24 27 30 33
27 0.149
30 0.06
33 0.009
37 0.478
Table 3: P-values of the Kolmogorov-Smirnov test between distributions of intervals between
consecutive RNA production events from PRM. For p-values <0.01, the hypothesis that the distributions are the same is rejected.
5
Single cell study of the dynamics of Repressilator
This chapter contains a brief introduction to the Repressilator, followed by a detailed description of the experimental and statistical methods used in the study. The Repressilator was chosen as a model for study as the dynamics of this genetic circuit is dependent on the activity of the promoters. The transcription kinetics of one of the promoters of the Repressilator has been described in Chapter 4 and it is found to be affected by temperature. In this chapter, we describe the effect of changes in temperature and copy-number on the dynamics of the Repressilator.