Difference between revisions of "Part:BBa K2627003"

Figure 1. CRISPRi system was induced by light. After the verification of the culture condition, we investigated the dynamic regulation effects of targeting gltA with light-controlled system through switching red light to green light at 2h, 5h and 9h. As shown in figure , all strains induced at different time grow better compared with illuminating at 0h. Bacteria growth was inhibited the most when being induced at 5h, showing an obvious palliation after sensing green light. The strains induced at the early-log phase (2h) grew slower than the control (upon red illumination), whereas it continuously grew and maintained a relatively high K value. It was supposed that cells growing at a moderate speed at early consumed less nutrition, thus drove the biomass constantly increase and had a higher level of K value. In accordance with the hypothesis, we concluded the negative control without crRNA targeting gltA which presented a lowest level of K value consumed too much nutrition so that it can’t satisfy the need of cell in late period of growth. In addition, inducing crRNA expression at 9h through green light, we found there were little growth variance between the strains green light induced at 9h and the control illuminated at red light continuously. It was supposed that at the stationary phase there was nearly no effect for inhibiting TCA cycle to repress the cell’s growth, which might due to its metabolic flow. The difference between the regulation effect of different induction time isn’t obvious through light switching compared with L-arabinose, might due to light sensor CcaS/CcaR system which only have 4.8-fold difference between red/green light induction.

Figure 2. CRISPRi system was induced by arabinose. With 0.2% L-arabinose induction, the samples showed an obvious repression on growth compared with the ones without L-arabinose (figure 2.). furthermore, we investigated the regulatory effects of targeting gltA at different stages of growth by adding L-arabinose at 0h, 2.5h, 5h, 7.5h and 9h. As shown in the figure, there presented an obvious difference between different induced time, in which the strains added L-arabinose at the beginning had the strongest repression in growth while with the delay of the induction time, the effect of inhibiting growth was gradually weakened. It indicated that this system can inhibit TCA cycle through inducing crRNA targeting gltA at different time, thus it proved that our idea that dynamically regulating metabolic flux was feasible to some extent.

Figure 3. Effects of copy number. To optimize the expression of crRNAs in EE-E15, then we constructed a medium-copy plasmid (pSR43.6) and high-copy plasmid (phz) to express crRNA targeting endogenous gltA. The two strains showed significant variance in growth in figure 4, suggesting crRNA on high-copy plasmid showed stronger repression than on medium-copy one. We supposed that the inhibition function was related to the quantity of crRNA and light-sensing promotor PcpG2-172 works well in high-copy number, leading to this result. It indicated that copy-number for light-induced crRNA had a great impact on this photoactivated regulation system.