For decades, it has been an everlasting and ambitious dream of "bioengineers" to optimize metabolic pathways, so as to maximize certain products of desire. However, despite being encouraged by this dream, scientists are invariably upset by the following problems they encounter during their attempts to regulate a pathway:
In order to regulate an entire pathway, it is necessary to control multiple genes. So -- Problem 1. How to simultaneously control the expression of SEVERAL genes in the pathway, but in a convenient way? Traditionally, researchers may use an independent inducing chemical respectively for each gene. But... But think about the boredom, the frustration of those laborious work of adding several chemicals, adjusting the amount, and keeping their concentration stable.
Some pathways are endogenous, or have been artificially implemented into the genome. So -- Problem 2. How to control genes that reside on the GENOME? If the genes are genomic, it would be much less feasible to substitute their promoters with those that can be adjusted by outside signals. eg. To place a genomic gene under the control of a promoter that is regulated by light sensors.
Often, people want to change their target. So -- Problem 3. How to quickly REDIRECT regulations onto other targets? If the researcher decides to regulate another gene, or to optimize another pathway in another project, it would be a repetition of the same work. eg. To reconstruct a similar plasmid, or to substitute the promoter of another genomic genes.
This year, our team, SJTU-BioX-Shanghai, is dedicated to solve these problems. We take the honor to present our product, the METABOLIC GEAR BOX. It is an easy-to-use BLACK BOX for our clients. And we have carefully evaluated our BOX to verify its function. See [http://igem.bio-x.cn/Team:SJTU-BioX-Shanghai/Results/Test/Overall Product Performance]. Our users need only care about:
1. The User Interface (UI) Design an Orthogonal Experiment, input the conditions of each group into the UI. These conditions can be set according to empirical values or dry-lab modelling (COBRA toolbox may be a good choice).
2. The Result Have a cup of coffee. Come back to gather your product, measure the amount, which would be a feedback to further experiment design.
How are those Problems Solved? Inside the Box, there are:
With the Metabolic Gear Box, here we present our --
Solution to Problem 1. To simultaneously control the expression of SEVERAL genes in the pathway, but in a convenient way Different Light Sensors are mostly independent. One light would only induce minimal response of another sensor. Our luminous system continuously produces stable lights. So unlike chemical regulation, researchers using our product do not have to wait to add additional chemicals to assure a stable concentration. Instead, take a rest, have a cup of coffee!
Solution to Problem 2. To control genes that reside on the GENOME Among a number of genome interference methods, CRISPRi, the newly developed tool, has standed out for its unparalleled simplicity (and thus, transferability), its relatively satisfying sequence-specificity and economical feasibility.
Solution to Problem 3. To quickly REDIRECT regulations onto other targets To target another gene, just change the 20bp base-pairing region of the guide RNA (gRNA), which determines most sequence specificity of CRISPRi. Replacing a 20bp segment -- that is all one have to do to redirect regulations! It is a VERSATILE platform!
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