Team:GreatBay China/Improve
mCATNIP2018@outlook.com
@Igem2018C
Improvement
BBa_J23119 is a classical strong consensus promoter that has been widely used among the iGEM community. This year, we designed a transcription-activator-like effector (TALE) stabilised version of J23119, TALE2 sp6, which is included as a member of the TALE stabilised promoter family. (Part Collection)
Figure 1. Design of TALE stabilised promoters. (a) TALE stabilised promoters can be considered to consist of two components: the genetic circuit encoding for TALE repressor and the core promoter that TALE represses. (b) Through an incoherent feedforward loop regulation, TALE stabilised promoters could achieve independence of gene expression from gene copy number.
Upon addition of a TALE2 genetic circuit, J23119 is engineered to become a core promoter.
Figure. 2 Differences in the sequence between J23119, UP119, and TALE2 sp6 core promoter
We first add an up-element which is an insulating spacer to separate it from the upstream TALE, creating an intermediate promoter UP119. Then the sequence between -35 region and -10 region of UP119 was altered to make a binding site for TALE2. The fluorescence characterisation results prove that the addition of UP119 on J23119 has no significant impact on its strength. The potential advantage of UP-element is that it separates the core promoter from other parts upstream, hence reducing the influence of those parts on the core promoter.
Figure. 3 Fluorescence characterization results show a distinct consistency of GFP expression regardless of change in copy number whereas J23119 and UP119 give rise in fluorescence at a higher copy.
The strength of TALE2 sp6 is remarkably lower than J23119, but has an almost constant transcription level on three different levels of copy number, since the transcription level of J23119 increases as the copy number increases. As a result, we have proven that TALE2 sp6 has significantly improved the expression consistency than J23119.