Bluprint of our project

It’s known that biological system is a rather complex collection of biodirectional processes in which the specific state of one object is broadly influenced by other closely associated objects. Can we simply change

Let’s take a food web for example. Apparently, the number of birds in a given wild is determined by both the plenty of worms and the number of its predators. In this way, are there proper ways to increase the number of birds? To untangle this question, factors like the feed strategy, water supply should be taken into consideration. Once optimized, factors as listed above may guarantee a relatively more suitable environmental for those birds to breed rapidly. However, inward restraints are usually the most fundamental factors. It’s obvious that a faster reproductive rate or a broader dietary contribute more to its final increase compared to factors like water supply.

Analogously, let’s take account of a newly found bacteria in which only finite promoters can be used to manipulate metabolic process. In this way, compared to outward temperature, concentration of oxygen, pH value or other factors, constraints from inward transcriptional processes are what we really need to bear in mind for an optimization of protein production. Nevertheless, as applied by temporal molecular labs, pure replacements of a stronger or weaker promoter may exceed the natural physiological limitations and ultimately end up in cell explosion.


Thus, a multi-level expression regulator wil make much sense towards a real optimization of production, which means it should be both adjustable and sequence-specific. In other words, an On or Off switch should be replaced by an adjustable slide rheostat-like knob switch to meet potential needs. Further on, we need two more novel modules, one of which is the contol panel and the other one is the knob. Then comes our project, entiled Master of Regulation: CRISPR/dcas9 & Tandem promoter-based Multi-level Gene Expression Regulator.

In our project, a tandem promoter system is used as the control panel. It’s reported that repetitive promoters have a relatively stronger expression level compared to a single promoter in question. Then we further develop a tandem promoter system, in which two of three disparately promoters are combined respectively. As a result, different expression levels become available, which means multi-level expression can be achieved in this way. For more details, please refer to our background part.

https://static.igem.org/mediawiki/2013/6/61/WHUTandemexamples.png" /> More importanly, a CRISPR/dCas9-based targeting system acts as the adjustable knob. In this system, the interaction between dCas9-gRNA and the DNA sequence of interest is highly spcific. Different expression level can be acquired when disparate promoter is targeted by dCas9-gRNA for either activation or repression. Consequently, combinations of diverse promoter transcriptional state finally lead to sequence-specific targeting and eventually an adjustable knob.