iGEM 2020 — Team Moscow

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Moscow/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Moscow/Engineering

[![](https://static.igem.org/mediawiki/2020/thumb/b/be/T--Moscow--logo-text.png/320px-T--Moscow--logo-text.png)](https://2020.igem.org/Team%3AMoscow)

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# Engineering success

## Overview

Last year, we conceived the concept of **split-system improvement.** However, while implementing the idea, our team met **several complications** with its purification. It turned out that a few other teams faced the same problem, so we made an attempt to discover why. We hypothesized that the **split-system might be too large** and, thus, complicated our work. During our further research, we found a **much smaller and relatively new protein - CasX**, which we used to design a new test system. To explore our device properties, we developed its **kinetic model** available in detail on our Modelling page. **Here, a comprehensive report on our molecular design, along with hardware, is represented.**

## Molecular design №1: split-system

##### Imagine and Design

Firstly, we decided to improve our previous year’s [(Moscow 2019)](https://2019.igem.org/Team%3AMoscow/Description#project-inspiration) design for HCV genotypes detection. This **system is supposed to work as follows**: two dCas9 proteins fused with N- and C-ends of β-lactamase bind to HCV’s cDNA target sequence. Binding results in the reunion of β-lactamase providing visible enzyme activity - oxidation reaction of nitrocefin. To determine the distance between two dCas9 proteins that will provide the most effective reunion of β-lactamase, **we made molecular modeling**.

##### Build and Test: Molecular modeling

We had the results of molecular modeling from the previous year, but **we decided to perform a more complex and full one**. Additionally, we already knew that PAM-out (PAM - protospacer adjacent motif, PAM-out - PAM regions are located on different DNA strands and oriented outward of the complex) orientation is more suitable for the split system [[1]](#1). This year **we simulated the structures of two dCas9 proteins in the nucleoprotein complex and calculated the optimal interval in nucleotides between them**. For this purpose, we wrote Python scripts for the Chimera to build models of two dCas9 proteins. To design these systems, we used the SpCas9 protein structure isolated from *Streptococcus pyogenes* (PDB id 5Y36)[[2]](#2). The structure of an ideal B-DNA was used for modeling the DNA separating dCas9 proteins. For PAM-out orientation, we generated models with different numbers of nucleotides between PAM regions. For choosing the best interval between Cas complexes, the graphs of the dependence of the distance between the geometric centers of the C-end, N-end, and the angles between them on the interval in nucleotides between the PAM regions were built (see Fig.1B). A model with the smallest angle and distance was chosen (the angle value was a priority when choosing a model), which also did not contain atomic overlaps between SpCas9 complexes, namely, the **model with an interval between PAM regions of 63 nucleotides** (see Fig.1B).

![Figure_1](https://static.igem.org/mediawiki/2020/2/23/T--Moscow--Figure_1_ES.png)

---

### References

1. Yihao Zhang et al., “Paired Design of DCas9 as a Systematic Platform for the Detection of Featured Nucleic Acid Sequences in Pathogenic Strains,” ACS Synthetic Biology 6, no. 2 (February 17, 2017): 211–16, https://doi.org/10.1021/acssynbio.6b00215.

   - Cong Huai et al., “Structural Insights into DNA Cleavage Activation of CRISPR-Cas9 System,” Nature Communications 8, no. 1 (November 9, 2017): 1375, https://doi.org/10.1038/s41467-017-01496-2.

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Moscow/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Moscow/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)