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Revision as of 12:55, 17 October 2018
Project Overview
Synthetic biology is now developing into its zenith time, and the concepts of BioBricks and standardized assembly methods have proven to be efficient, universally compatible and highly extensible. This year, Team SCU_China plans to bring the standardization in synthetic biology to a completely new level.
Overview
Huge scale of our experimental design based firmly on modelling results. Our Model can be divided into two parts, each has significant function in guiding the direction of our whole design, and the future work. Inspired by the modelling results, our team can optimize the experiment without wasting our time. Besides, the results help us to make the system stable and feasible.
Mismatch
Why we did this model?
1) The gradient regulation just based on some experiments focusing on the efficiency of sgRNA. No essay shows particular results and specialized experiment about the idea. It’s quite risky for us to have a try, no matter how fascinating the idea is. As a result, we should confirm its feasibility at first.
2) So many factors will influence the binding between the sgRNA-dCas9 complex with targeted DNA region. The hydrogen bound between bases, the specificity of sgRNA, the three-dimension structure of sgRNA scaffold. Before we start our experiment, we should consider all likely elements which have an effect on the results as thorough as possible. In order to save time, and generalize all the condition, the model stands out to be a perfect method.
3) The construction of mismatch sequence library is a huge work. With only two months to finish our project, it does not look like pragmatic to verify the idea by experiment.
2) So many factors will influence the binding between the sgRNA-dCas9 complex with targeted DNA region. The hydrogen bound between bases, the specificity of sgRNA, the three-dimension structure of sgRNA scaffold. Before we start our experiment, we should consider all likely elements which have an effect on the results as thorough as possible. In order to save time, and generalize all the condition, the model stands out to be a perfect method.
3) The construction of mismatch sequence library is a huge work. With only two months to finish our project, it does not look like pragmatic to verify the idea by experiment.
Figure 2. dCas9-mediated repression in E. coli.
The sgRNA directs dCas9 to promoter or open reading frame regions to prevent RNA polymerase binding or elongation, respectively
Orthogonality
Inspired by the modularization and “Do not re-invent the wheel” philosophy in computer programming, this year SCU_China iGEM team came up with the idea of using the dCas9 protein to manipulate the expression of proteins and implement complex logic in a single E. coli cell to achieve the similar “call-and-return” controlling paradigm.
Moreover, we want to construct versatile “library strains” that contains many, and maybe redundant coding sequences of commonly used proteins (like enzymes for industrial production etc.) which are dormant by default; such sequences could be on a large plasmid transformed in advance or integrated into the genome. When we need to use the function of particular proteins whose coding sequences are already exist in the cell (to “call” them), we just simply transform a much smaller (compared to the CDS) “Minimid” containing expression cassettes of sgDNAs targeting the desired proteins into the library strain, and then the dCas9-sgRNA complex would initiate/inhibit the transcription and then expression of the proteins of interest.
This way, we could not only simplify the construction of plasmids (for it is small and could even be directly synthesized), but also provide a rapid way of constructing different strains for production and a possible method to avoid genetic pollution. What’s more, utilizing dCas9 system also enables us to implement complex logics in one cell. Our prospect is that using the system, finally we could really literally “program” the cell, i.e. using computer language to code the logic, and then use a “genetic compiler” to convert the logic to nucleotide sequences (for “Minimid”) containing a series of well-designed sgDNAs.
Moreover, we want to construct versatile “library strains” that contains many, and maybe redundant coding sequences of commonly used proteins (like enzymes for industrial production etc.) which are dormant by default; such sequences could be on a large plasmid transformed in advance or integrated into the genome. When we need to use the function of particular proteins whose coding sequences are already exist in the cell (to “call” them), we just simply transform a much smaller (compared to the CDS) “Minimid” containing expression cassettes of sgDNAs targeting the desired proteins into the library strain, and then the dCas9-sgRNA complex would initiate/inhibit the transcription and then expression of the proteins of interest.
This way, we could not only simplify the construction of plasmids (for it is small and could even be directly synthesized), but also provide a rapid way of constructing different strains for production and a possible method to avoid genetic pollution. What’s more, utilizing dCas9 system also enables us to implement complex logics in one cell. Our prospect is that using the system, finally we could really literally “program” the cell, i.e. using computer language to code the logic, and then use a “genetic compiler” to convert the logic to nucleotide sequences (for “Minimid”) containing a series of well-designed sgDNAs.
Indigo synthesis