CRISPRi-on

At present, our Metabolic Gear Box, which combines CRISPRi with light sensors, only down-regulates three genes of a pathway. However, in many other cases, genes in a synthetic pathway are supposed to be up-regulated in order to acquire the most products.
A newly devised tool, CRISPR-on, has provided some inception. CRISPR-on devisers simply fuse a transcriptional activation domain with dCas9 to create a sequence-specific transcription activating tool. CRISPR-on has been proved to be effective in human and mouse. We expect to conduct similar work in our bio-factory, Escherichia coli.
Our plan is to fuse alpha factors with dCas9 protein, which has previously been proved to be a successful method to create blue-light-induced transcription factors (Camsund et al., 2011). Our design is shown below.

However, to integrate CRISPRi and CRISPR-on would never be an easy task, since sgRNA for CRISPR-on is supposed to target upstream of promoter, rendering it necessary to incorporate logical switches.

Smaller Light Sensor

The RGB light sensors we currently utilize are all large systems, with proteins consisting of some five-hundred amino acids. Therefore, it would help simplify our system a lot if there are small sensors. The smallest light sensors are no doubt those light-induced dimerizing transcription factors (Camsund et al., 2011), containing no more than one-hundred amino acids. Furthermore, these direct sensors has claimed to be extremely sensitive.

Unfortunately, all reported small sensors in E. coli senses blue light. So we would attempt on creating tiny sensors for red and green light.

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