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Revision as of 03:07, 28 September 2013
PROJECT
Multicellular Analog Clock
Summary
Our concept of the multicellular analog clock is based on qualitative assumption such as how negative feedback loop behaves, how AHL diffuses, and so on. To ascertain our multicellular analog clock can function as an analogue clock, namely, to confirm the feasibility of our cell-cell communication included gene circuit, and to deepen understanding of behavior of the system, we conducted the following simulation.
Our model for multicellular analog clock consists of four parts: DDE analysis, parameter sensitivity analysis, parameter sweep, stochastic analysis. DDE analysis is to examine the feasibility of our project, and also provides the foundation for the other parts of analysis. Through parameter sensitivity analysis, we gained more insight of the relationship between input and output variables. The insight led to the third part of analysis, in which parameter sweep enabled us to grasp appropriate ranges fo the identified sensitive parameters. Finally, we conducted stochastic analysis with the sensitive parameters fixed, and simulate our device's behavior under the actual conditions.
Background
To simulate the cell-cell communication system, we developed a delayed differential equation model. The equation used in the model are followings. The variables are described in the following table.
In our cell-cell communication system, the major kinetic events are: mCherry synthesis and degradation, LuxI synthesis and degradation, TetR synthesis and degradation, AHL synthesis and degradation. These kinetic events are contained in the equations. The following describes how the equations are developed.
- mCherry synthesis and degradation
- LuxI synthesis and degradation
- TetR synthesis and degradation
- AHL synthesis and degradation
Systems
Results and Discussion
References
RNA Silencing
Summary
Our concept of the multicellular analog clock is based on qualitative assumption such as how negative feedback loop behaves, how AHL diffuses, and so on. To ascertain our multicellular analog clock can function as an analogue clock, namely, to confirm the feasibility of our cell-cell communication included gene circuit, and to deepen understanding of behavior of the system, we conducted the following simulation.
Our model for multicellular analog clock consists of four parts: DDE analysis, parameter sensitivity analysis, parameter sweep, stochastic analysis. DDE analysis is to examine the feasibility of our project, and also provides the foundation for the other parts of analysis. Through parameter sensitivity analysis, we gained more insight of the relationship between input and output variables. The insight led to the third part of analysis, in which parameter sweep enabled us to grasp appropriate ranges fo the identified sensitive parameters. Finally, we conducted stochastic analysis with the sensitive parameters fixed, and simulate our device's behavior under the actual conditions.
Background
Introduction
It is needless to say that gene knockdown is important in the study of biology. Genomic knockdown by making the target gene mutate requires much labor and time. In eucaryotes, RNAi has contributed to the functional analysis of genes. E.coli, which belongs to procaryotes, does not have the mechanism needed for RNAi and this method is not usually used for E.coli.
But in recent years it has been becoming clear that E.coli has the mechanism of gene silencing by small regulatory RNAs (sRNA) which have complementary sequences with their target mRNAs [1].We used the mechanism for gene knockdown by designing synthetic sRNAs and inducing plasmids coding them into bacterial cells. This method can be done by gene introduction and enables us to knock down genes by combining our new BioBrick parts "micC scaffold" and complementary sequences with the target gene [2].
We tested using our BioBrick parts by applying them for the following objectives.
- Acceleration of Biosynthesis of L-DOPA, a substance utilized for treatment of Parkinson’s disease.
- Improvement of H2 Productive Capacity of E.coli
- Suppressor of Multi-Negative Feedback System of Clock Device
System
Mechanism of RNA silencing in Escherichia coli
Escherichia coli have several trans-acting sRNA molecules repressing the expressions of their target genes, such as micC and sgrS and so on. Their mechanism has been becoming clear in recent years. (Fig.1) These sRNA molecules consint of complementary sequences to their target mRNAs and a scaffold sequence that recruites the Hfq protein. Hfq protein, one of the RNA chaperones, facilitates the hybridization of sRNAs to mRNA by their complementary sequences and with RNase E. RNase E causes the degradation of the sRNAs by the target mRNAs. So the mechanism of gene repression by Hfq-dependent sRNAs is composed of the translational repression by their complementary sequences and Hfq proteins and the degradation of the target mRNAs by RNase E [3].
We tried to apply the Hfq protein-dependent RNA silencing for several objectives by establishing the design principle of sRNAs.
Fig.1 Mechanism of expression inhibition by Hfq protein-dependent sRNAs Design and use of synthetic regulatory small RNAs
In order to design Hfq protein-dependent sRNAs simply, we used the following design principle. Our sRNAs are composed of two regions: a target-binding sequence and a scaffold sequence. We made the sRNA generator by combining these with a promoter and a terminator. (Fig.2)
We chose a region of the target sequence that spans the AUG to nucleotide +21 of the mRNA as the binding site. This was because a previous study suggested that it is effective especially to select the region overlapping the TIR (Translation Initiation Region) of the mRNA [2]. The scaffold sequence recruits the Hfq protein, which facilitates the hybridization of sRNA and target mRNA as well as mRNA degradation. Of the scaffold sequences of E.coli, we selected MicC because of the superior repression capability [2].
Fig.2 Design principle of our synthetic sRNAs
Automatic RNA silencing primer designer
CDS
Forward Primer for BioBrick
Forward Primer for Inverse PCR
Reverse Primer for Inverse PCR
Application
Acceleration of Biosynthesis of L-DOPA
n order to make E.coli produce L-DOPA, we made hpaBC (encoding the enzyme 4-hydroxyphenylacetate 3-hydroxylase) BioBrick parts by cloning it from E.coli BL21 strain. HpaBC catalyzes the hydroxylation of tyrosine and produces L-DOPA (Fig.3a). But the production rate of L-DOPA was low. Then we designed sRNAs knocking down several genes involved in the pathway from glucose to L-Tyr in order to increase the carbon flow to tyrosine synthetic pathway, because the amount is thought to be dependent to that of L-Tyr, the precursor of L-DOPA [4].
We selected tyrR(encoding tyrosine repressor), csrA(encoding carbon-storage regulator, which regulates the expression of enzyme genes involved in glycolysis) and tyrC (phosphoribosylanthranilate isomerase) as the targets [2]. tyrR and csrA have been shown to promote L-Tyr biosynthesis in a previous study.(Fig.3b)
Because these sRNA are thought to damage the growth of E.coli, we tried several promoters having different strength.
Fig.3a Hydroxilation of tyrosine to L-DOPA by HpaBC
Fig.3b The tyrosine biosynthetic pathway in E.coli and our metabolic engineering strategy
Improvement of H2 Productive Capacity of E.coli
In 2012, we UT-Tokyo made "H2 E.coli", which produces more H2 than usual E.coli. This year we tried to improve its H2 productive capacity further by designing anti-hycA.
E.coli naturally produces hydrogen through a process called mixed acid fermentation (MAF pathway) (Fig.4a). HycA represses the transcription of the proteins of FHL complex, which produces H2 from formate.
Last year we tried to derepress it by introducing plasmids including many decoy binding sites in our project "Inhibition without knockdown". This method has several methods. It allows to knockdown by introducing BioBrick parts and does not make scars on the candidate's genome. But it can be used only for DNA binding proteins and the strength cannot be regulated without changing the plasmid.
Though the merit of "Inhibition without knockdown" is retained in knockdown with Hfq protein-dependent sRNAs, our new method does not have these demerits.
Fig.4a The function of FHL complex and HycA
Fig.4b The system of "Inhibition without knockout"
By introducing plasmids with many decoy binding sites of the target proteins, we tried to prevent repressors from acting on genomic sites.
Suppressor of Multi-Negative Feedback System of Clock Devise
In our project, Multicellular Analog Clock, we needed a suppressor to be used for the Multi-Negative Feedback System and UV reset devise. Our clock devise uses novel systems which are not seen in previous papers or past iGEM project and it is difficult to say what kind of suppressors is best unless we actually try them. So we designed two kinds of sRNAs that bind to the TIR of mCherry and luxI as the suppressors (anti-mCherry and anti-luxI).
sRNA suppressors will behave differently from AiiA and TetR. sRNAs express and decompose more rapidly than these protein based suppresors and act on different targets.
The two sRNAs are act on different molecules within the clock pathway and therefore expected to influence clock behavior differently. Translational inhibition is caused only by sRNAs binding to the TIR of the target gene while degradation by RNase E is caused by any sRNAs binding to the mRNA.
The differences of mechanisms may lead to various patterns of suppression of various strength and time.
References
[1]Aiba, H. (2007). Mechanism of RNA silencing by Hfq-binding small RNAs. Current opinion in microbiology, 10(2), 134-139.
[2]Na, D., Yoo, S. M., Chung, H., Park, H., Park, J. H., & Lee, S. Y. (2013). Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nature biotechnology.
[3]Yoo, S. M., Na, D., & Lee, S. Y. (2013). Design and use of synthetic regulatory small RNAs to control gene expression in Escherichia coli. Nature protocols, 8(9), 1694-1707.
[4]Munoz, A. J., Hernandez-Chavez, G., de Anda, R., Martinez, A., Bolivar, F., & Gosset, G. (2011). Metabolic engineering of Escherichia coli for improving L-3, 4-dihydroxyphenylalanine (L-DOPA) synthesis from glucose. Journal of industrial microbiology & biotechnology, 38(11), 1845-1852.
[5]Maeda, T., Sanchez‐Torres, V., & Wood, T. K. (2008). Metabolic engineering to enhance bacterial hydrogen production. Microbial Biotechnology, 1(1), 30-39.
