Abstract
The models for the Repressilator and the Toggle Switch that we have built in Chap. 8 are not based on the notion of DNA parts and the two circuits are treated just as biological systems made of interacting molecules. However, as we have seen in Chap. 1 , an ultimate goal of Synthetic Biology is to developed methods for the modular design and modeling of genetic circuits, where basic components – DNA sequences and pools of signal carriers – are linked together in a visual way, as it is usually done with electronic circuits. One of the first attempts in this direction is represented by the Parts & Pools software, the Synthetic Biology add-on of ProMoT, a computational tool for the modular design and analysis of complex systems. In the following, we will see how the Repressilator and the Toggle Switch can be designed in a “drag and drop” way with Parts & Pools. First, DNA parts and pools are displayed on the canvas provided by ProMoT. Then, they are connected to each other with wires were the signal carrier molecules are imagined to flow. Each circuit component is associated with a pre-existent mathematical model, in which a user can only change parameter values. A model for the whole circuit is generated by ProMoT via the composition of the models of the circuit components. The circuit model can be exported, finally, into the SBML format and used for simulations and analysis with COPASI.
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Notes
- 1.
ProMoT is freely available at http://www2.mpi-magdeburg.mpg.de/projects/promot/ .
- 2.
Despite the fact that RNA polymerase is an enzyme, in the Michaelis-Menten scheme that describes the interaction between RNA polymerase and promoter, the promoter plays the role of the enzyme E and the RNA polymerase that of the substrate S – see Eq. (2.46).
- 3.
Also in this case the ribosomes are the substrate for the Michaelis-Menten scheme and the mRNA corresponds to the reaction enzyme.
- 4.
PoPS in corresponds to a PoPS out from a terminator.
- 5.
Wherever possible, we will use the same parameter values as in Chap. 8 .
- 6.
Here we use ProMoT version 0.8.5 on Linux (Ubuntu) operating system. To launch ProMoT graphical user interface, you should run the script “promot-ui” located in the directory “/usr/local/promot/scripts/” if you have made a default ProMoT installation.
- 7.
By clicking on “…” on the left of each entry in this window you can browse the directories in your computer.
- 8.
If you prefer, you can copy all Perl scripts, input files, and icon files (PNG) from “/usr/local/promot/kb/scripts/synthetic-biology/” to the “repressilator” directory and run the perl scripts from a terminal.
- 9.
A more recent version of Parts & Pools – developed mainly for eukaryotic gene circuit design [36] – does not put any limit on the operator number.
- 10.
Each string or number you write into a Parts & Pools input file shall not be included between quotation marks nor apices. Moreover, it must be separated by a blank space from the semicolon on its left.
- 11.
If, like in this case, the same transcription factor binds both operators, its name should be followed by the number 1.
- 12.
Readthrough means that RNA polymerase molecules do not leave the DNA after meeting a transcription termination signal but flow into an adjacent promoter without ceasing mRNA production.
- 13.
MDL used d instead of e to represent powers of 10.
- 14.
“alpha1f” and “alpha1t” would have the same value.
- 15.
If rate constant values are not consistent with cooperative effects, the “promoter_gen.pl” script returns a warning message.
- 16.
In principle, it holds that k 1sa > k 1, k −1sa < k −1, and k 2sa > k 2. When these conditions are not respected only a warning message is returned by the “promoter_gen.pl” script.
- 17.
Here we call pseudo-polycistronic a multicistronic sequence where DNA spacers have been omitted.
- 18.
The Parts & Pools modeling framework supposes that every post-transcriptional regulation takes place inside the RBS. This assumption does not provoke any loss of information even though, in living cells, small RNA molecules can be complementary to portions of the CDS or the 3’UTR.
- 19.
This value comes from the assumption that the mean RBS length is of 20 nucleotides and the average RNA polymerase elongation speed in E. coli equals 40 nt/s [28].
- 20.
- 21.
In COPASI, we will change the initial concentration of one repressor to 10−8 M, as in Chap. 8 .
- 22.
It is useful, for future circuit modifications, to save into a file all modules you used for designing the circuit. Click on “File, Save All Classes…” and name the file as “ALL_repressilator.mdl”.
- 23.
A version of this script for MS Windows is also available.
- 24.
If you are using a terminal, type “/usr/local/promot/kb/scripts/synthetic-biology/parser_lev2_linux.pl repressilator”.
- 25.
This indeed is the number of the equations calculated by ProMoT when the Repressilator model was exported into the SBML format.
- 26.
In this version of Parts & Pools the interactions between chemicals and transcription factors take place only inside promoter parts.
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Marchisio, M.A. (2018). Computational Gene Circuit Design. In: Introduction to Synthetic Biology. Learning Materials in Biosciences. Springer, Singapore. https://doi.org/10.1007/978-981-10-8752-3_9
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