Team:WHU-China/templates/standardpage introduction

From 2013.igem.org

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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. </br></br>
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If people want to drive a system to work for them, even if the system is a simple one, simply keep pushing the system toward the goal may not be the best shoot. For example, if a professor is too pushing, his students may, on the contrary, unable to perform their best; if a farmer adding too much fertilizer, the land may be damaged in the long run, etc.</br></br>
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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. </br></br>
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Biological systems are extremely complex, and the components in the system are intensely interconnected. So in order to exploit the maximum potentiality of a biological system, we'll have to keep the protein or metabolic product production in a desired range. Not too high, as it may hurt the cell or inhibit its growth; either not too low, as it will be economically inefficient. </br></br>
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<img src="https://static.igem.org/mediawiki/2013/e/e9/WHUswitches.png" align=right /></br>
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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. </br></br>
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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. </br></br>
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So how can we reach a desired range of expression? We need to properly combine the transcription and translation initiation elements, just as an recent published Nature article suggested[1]. But that paper just used the throughly studied E.coli expression elements in E.coli. What if we are doing engineering in a non-model organism that we just have data about a handful of expression elements, can we create the elements we need? </br></br>
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Our project proposed a way to employ a limited set of promoters to reach any desired expression level, or even switch between several expression level. </br></br>
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<img src="https://static.igem.org/mediawiki/2013/9/95/WHUIntro1.png" /></br></br>
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First, we combine the known promoter into tandem promoter system. We've done experiments and modeling to show how can we use a 0.1 promoter and a 0.3 promoter to reach expression level from 0.1 to the maximum. Please check experiment here and modeling here. </br></br>
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<img src="https://static.igem.org/mediawiki/2013/6/6f/WHUIntro2.png" /></br></br>
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<img src="slide" /></br></br>
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Then, we make the tandem promoter a control panel by using d/aCas9 to regulate it. This enable the tandem promoter to switch between several designable expression level. This is different from the normal regulated promoter, which usually has only two stage: on and off. Our multistage promoter can serve as a universal solution for real-time precise regulation of target gene. To see our experiment about this multistage promoter, please click here. It's also important to ensure the orthogonality of this multistage promoter. So the off-target tendency of Cas9 is modeled and analyzed by combining the data of six paper about Cas9 off-target. For the modeling result, please click here. </br></br>
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<img src="https://static.igem.org/mediawiki/2013/e/ef/WHUTandemPro2.png" style="width:500px;height:auto;" /></br>
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<img src="https://static.igem.org/mediawiki/2013/a/a8/WHUSwitches.png" /></br>
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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.
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Revision as of 21:50, 27 September 2013

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


If people want to drive a system to work for them, even if the system is a simple one, simply keep pushing the system toward the goal may not be the best shoot. For example, if a professor is too pushing, his students may, on the contrary, unable to perform their best; if a farmer adding too much fertilizer, the land may be damaged in the long run, etc.

Biological systems are extremely complex, and the components in the system are intensely interconnected. So in order to exploit the maximum potentiality of a biological system, we'll have to keep the protein or metabolic product production in a desired range. Not too high, as it may hurt the cell or inhibit its growth; either not too low, as it will be economically inefficient.

So how can we reach a desired range of expression? We need to properly combine the transcription and translation initiation elements, just as an recent published Nature article suggested[1]. But that paper just used the throughly studied E.coli expression elements in E.coli. What if we are doing engineering in a non-model organism that we just have data about a handful of expression elements, can we create the elements we need?

Our project proposed a way to employ a limited set of promoters to reach any desired expression level, or even switch between several expression level.



First, we combine the known promoter into tandem promoter system. We've done experiments and modeling to show how can we use a 0.1 promoter and a 0.3 promoter to reach expression level from 0.1 to the maximum. Please check experiment here and modeling here.





Then, we make the tandem promoter a control panel by using d/aCas9 to regulate it. This enable the tandem promoter to switch between several designable expression level. This is different from the normal regulated promoter, which usually has only two stage: on and off. Our multistage promoter can serve as a universal solution for real-time precise regulation of target gene. To see our experiment about this multistage promoter, please click here. It's also important to ensure the orthogonality of this multistage promoter. So the off-target tendency of Cas9 is modeled and analyzed by combining the data of six paper about Cas9 off-target. For the modeling result, please click here.


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