3D modeling

The structure of the original LEAP2 was taken from the PDB database (PDB id: 2L1Q). 3D structures of chimeric proteins were obtained using trRosetta tool with default parameters

The Leap2 protein and chimeric proteins 3D structures./center>

Using obtained 3D structures, we performed 3D alignment of chimeric proteins with original Leap2 protein in PyMol software using align plugin (fig. 4). We assume that the addition of Myc-tag and LMWP to the C-terminus of the LEAP2 protein did not affect deleteriously the 3D structure and the N-terminal region retained the ability to bind the Ghrelin receptor.

The 3D structure alignment of chimeric proteins (left – LEAP2/Myc-tag, right – LEAP2/Myc-tag/LMWP) with the original LEAP2 protein.

Codon optimization

To get a better expression of our chimeric protein in the bacterium Escherichia coli strain K12 codon optimization of nucleotide sequences was performed using J-cat tool.

Also, VaxiJen server was recruited to predict the immunogenicity of the chimeric proteins. Overall prediction for the protective antigen was equal to 0.348. This indicates that our chimeric proteins are non-antigen.

Genetic oscillator


Before talking about the creation and modelling of a genetic oscillator, it is necessary to understand what it even is. An oscillating gene is a gene whose expression changes periodically over time. Accordingly, genetic oscillators are cell signalling systems that contain oscillating genes, and therefore, these are systems whose state periodically changes over time [1].

Genetic oscillators are very common among both prokaryotes and eukaryotes - for example, circadian rhythms, which are the most popular example of a genetic oscillator, are even found in cyanobacteria, and we encounter circadian rhythms in plants and animals every day (and night). The design and modelling of genetic oscillators is a flourishing field of synthetic biology, as it is a good way to regulate gene expression to achieve rhythmic increase and reduction over time. For our project, we need such a system to regulate the gene LEAP2, an inverse agonist of ghrelin.

How do we design a genetic oscillator?

An important parameter in models of genetic oscillators is the period, which is the period of time our system takes to make one oscillation. In our model, we need the bacterium to express the gene LEAP2 approximately 4 times a day which means that the oscillation period of our genetic oscillator should be about 6 hours.

Since the creation of the first genetic oscillator (in the 2000s), several types of oscillators have been designed, of which we chose the following for further work:

  • Repressilators
  • Oscillators based on circadian rhythms

Below we will tell you about the features of each selected type:

The repressilator

A repressilator is a system of several genes in which the product of each gene inhibits the synthesis of a subsequent gene. A diagram of a 3 gene repressilator is shown in the picture below [2].

The oscillation frequency of such a model depends on the number of genes in it (the more genes, the longer the period) and on the lifetime of the product of each gene. For example, the period of oscillations can be significantly reduced if we add sites recognized by proteases to the products of genes A, B, and C, since then proteins will degrade much faster.

A three-genes repressilator

Why is this particular model appealing to us?

Mathematical models show that a repressilator model of n genes, where n is an odd number, will work [3]. This means that by adding genes we can achieve the desired oscillation period.

What are the disadvantages of this model?

From the literature, we know that the oscillation period is calculated in minutes, but not hours, so if we test a repressilator model of n genes with a suitable period, serious problems may arise with its implementation in cells.

Currently, one of the fronts of our team's work is to figure out how many genes are needed and what additional parameters should be taken into account for modelling an oscillator with the required oscillation period.

An oscillator based on circadian rhythm

Circadian rhythms are rhythms with an interval of about 24 hours, which are found, as mentioned earlier, in both eukaryotes and prokaryotes, which makes it possible for us to implement such a pattern in our bacteria.

Perioad of a circadian oscillator

Why is this particular model appealing to us?

This model has a very long period of oscillations, so our task is not to increase the period of oscillations to 6 hours, as in the case of a repressilator, but to reduce it. The genetic oscillator responsible for circadian rhythms in cyanobacteria can be used as the basis.

Also, we already know to some extent which parameters influence this model. For example, research has already been carried out [4] on how certain parameters can affect the stability of the oscillation of the cyanobacterial circadian clock, in particular, the ratio of the two proteins KaiA and KaiC that make up this system.

In addition, attempts have already been made to transfer this oscillator into E.coli bacteria by other iGEM teams [5], so we now know some existing methods of implementation of this model.

What are the potential disadvantages of this model?

The cyanobacterial "circadian clock" is much more complex than the repressilator, there are many concurrent processes and many parameters to consider, which are difficult to model. The second front of our work is devoted to the study of how the oscillator already existing in cyanobacteria can be adapted to our task.

What are our plans for the future

We plan to continue working in two directions, trying to create a working model based on both the repressilator and the circadian rhythms of prokaryotes. After that, we plan to engage in the implementation of the resulting model and in vivo testing in order to assess whether the resulting system really meets our requirements.


  1. Kruse K, Jülicher F. Oscillations in cell biology. Curr Opin Cell Biol. 2005 Feb;17(1):20-6. doi: 10.1016/ PMID: 15661515.
  2. Purcell O, Savery NJ, Grierson CS, di Bernardo M. A comparative analysis of synthetic genetic oscillators. J R Soc Interface. 2010 Nov 6;7(52):1503-24. doi: 10.1098/rsif.2010.0183. Epub 2010 Jun 30. PMID: 20591848; PMCID: PMC2988261.
  3. Strelkowa N, Barahona M. Switchable genetic oscillator operating in quasi-stable mode. J R Soc Interface. 2010 Jul 6;7(48):1071-82. doi: 10.1098/rsif.2009.0487. Epub 2010 Jan 22. PMID: 20097721; PMCID: PMC2880078.
  4. Nakajima M, Ito H, Kondo T. In vitro regulation of circadian phosphorylation rhythm of cyanobacterial clock protein KaiC by KaiA and KaiB. FEBS Lett. 2010 Mar 5;584(5):898-902. doi: 10.1016/j.febslet.2010.01.016. Epub 2010 Jan 16. PMID: 20079736.
  5. UChicago 2015 iGEM Team. KaiABC Oscillator: