iGEM 2021 — Team Moscow_City

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# Team:Moscow City/Notebook

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# 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)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Moscow City/Engineering

<!DOCTYPE html>

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* [Home](https://2021.igem.org/Team%3AMoscow_City)
* Project

  + [Description](https://2021.igem.org/Team%3AMoscow_City/Description)
  + [EHV](https://2021.igem.org/Team%3AMoscow_City/EHV)
  + [Contribution](https://2021.igem.org/Team%3AMoscow_City/Contribution)
* [Human Practices](https://2021.igem.org/Team%3AMoscow_City/Human_Practices)

  + [Human Practices](https://2021.igem.org/Team%3AMoscow_City/Human_Practices)
  + [Collaborations](https://2021.igem.org/Team%3AMoscow_City/Collaborations)
  + [Implementation](https://2021.igem.org/Team%3AMoscow_City/Implementation)
  + [Communication](https://2021.igem.org/Team%3AMoscow_City/Communication)
* [Attributions](https://2021.igem.org/Team%3AMoscow_City/Attributions)
* [Engineering](https://2021.igem.org/Team%3AMoscow_City/Engineering)

  + [Engineering](https://2021.igem.org/Team%3AMoscow_City/Engineering)
  + [Lab Book](https://2021.igem.org/Team%3AMoscow_City/Lab_book)
  + [Model](https://2021.igem.org/Team%3AMoscow_City/Model)

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Engineering success

Design

![](https://static.igem.org/mediawiki/2021/7/7a/T--Moscow_City--tild3234-6138-4535-a633-633337356632_f1.png)

![](https://static.igem.org/mediawiki/2021/f/f3/T--Moscow_City--tild3762-3838-4137-b262-303234663136_f2.png)

[Dry lab design](https://static.igem.org/mediawiki/2021/e/e1/T--Moscow_City--gRNA.pdf)

**We have decided to check two potentially suitable systems:**

System design

**Intein system:**
When two proteins get closer due to landing on viral RNA, trans-splicing happens, and peptide bond forms between beta-lactamase halves with also forming an active enzyme. It cleaves nitrocefin and the appearance of red coloration indicates the presence of the viral RNA in the solution.

Left

Right

1. Scheme of the intein construct
2. Scheme of trans-splicing

**For the system without intein:**
Full beta-lactamase forms when two proteins get closer due to landing on viral RNA. It splits nitrocefin, and the appearance of the red color signals us that there is a viral RNA in our solution.

1. Scheme of the non-intein construct

**Design:** When choosing between different Cas-proteins, we decided to work with LwadCas13a protein capable of detecting RNA because LwadCas13a was already available in our host lab, so we focused on our initial gRNA and protein design around it.

We chose beta-lactamase as a reporter protein because it is well-known and reliable. We also had it encoded in AmpR in our lab.

We also understood that we need to place beta-lactamase fragments far from dcas13a to not interfere in each other’s functions. Therefore, we added a medium-sized flexible linker consisting of neutral amino acids: GGGGGG.

**You can read more about systems choosing here:**
[**BBa\_K4088888**](http://parts.igem.org/Part%3ABBa_K4088888)**,** [**BBa\_K4088889**](http://parts.igem.org/Part%3ABBa_K4088889)**,** [**BBa\_K4088890**](http://parts.igem.org/Part%3ABBa_K4088890)**,** [**BBa\_K4088891**](http://parts.igem.org/Part%3ABBa_K4088891)**.**

During the **modeling of our experiment**, we have been choosing between two ways of cloning: sequential ligation and Golden Gate.

We chose **Golden Gate** for dCas13 and beta-lactamase fragments insertion. Golden Gate does cloning: to introduce multiple inserts - thanks to which one does not have to do screening after each insertion, which saves development time. In addition, with linear ligation, the yield is negligible due to the inefficiency of ligation and the small amounts of DNA extracted from the gel. **Thus, we increase the yield of DNA using the Golden Gate.**

**Build:** We will use Golden Gate cloning to assemble our both systems. For the intein constructs, the intein fragments will be added by restriction/ligation at sites close to the AmpR gene fragment (see the Build section for details).

**In the end, we will get genes of each chimeric protein.** We will express dcas13a-C\_lact and dcas13a-N\_lact from pet-28(+) и pet-30(+) plasmids due to the convenient location of His-tags and restriction sites. Also, the pET system is the most powerful system yet developed for the cloning and expression of recombinant proteins in E. coli. We will also express similar chimeric proteins with intein hanging fragments dcas13a-C\_lact-C\_int and dcas13a-N\_lact-N\_int. All four proteins will be expressed in an expression culture that is often used in our lab, Rosetta (suitable for protein expression with pET).

In addition, we simulate the interaction of beta-lactamase fragments and intein fragments for a preliminary comparison of the efficiency of our systems.

**Test:** During the experiments, we will test the efficiency of both systems by comparing the time it took solutions with different protein systems to change their staining intensity to some fixed value using a multi-well spectrophotometer. If the binding efficiency of the beta lactamase fragments without split inteins is low, we may not wait for a change in staining - and then there will be false negatives. Therefore, we will also set up a real time PCR to detect the RNA of the virus in solutions. Thanks to the data obtained, we will make a conclusion about the efficiency of the detection systems used. We will also use several controls (more details in the Test section)

**Learn: In the end, we will choose the best detection system.**
Our goal is a test strip for virus detection. We also want to make the test sufficiently accurate and as fast as possible - for this, we use HUDSON so that we don't have to perform RNA extraction. However, we will start with a plate with a freshly prepared chimeric protein solution with nitrocefin in the wells, to which we will add the RNA of the virus after LAMP (loop-mediated isothermal amplification). We also aim at the fact that the use of our test will not involve the use of spectrophotometry, but at the stage of development, it should be used for comparison, calibration of the systems.

Build

See our lab book page
mOdeling page

**Primers were selected using SnapGene.**
**﻿**We checked Melting temperatures in the NEB Tm Calculator online software.

![](https://static.igem.org/mediawiki/2021/d/d4/T--Moscow_City--tild3139-3139-4630-b431-373737323662_press_f.png)

In SnapGene, we modeled molecular cloning of four plasmid genetic constructs producing fusion protein. They included four plasmid assemblies.Two of them contained: the N- or C-terminal half of the beta-lactamase gene combined with the catalytically inactive dCas13a using a polyglycine linker. The other two had the same construct, including the N- or C-terminal half of the intein together with the N- or C-terminal half of the beta-lactamase, respectively.

**Download SnapGene history**

[Snapgene](https://static.igem.org/mediawiki/2021/8/89/T--Moscow_City--snapgene_engineering.pdf)

In the lab, we have started to build our system: we have now successfully cloned the dcas13a-C\_lact gene. See the lab book for more details

[Lab protocols](https://static.igem.org/mediawiki/2021/a/a3/T--Moscow_City--Lab_Protocols_%282%29.pdf)

[Amino acid sequence and Molecular mass](https://static.igem.org/mediawiki/2021/f/f1/T--Moscow_City--Amino_acid_sequence_%282%29.pdf)

[Troubleshooting on Protocols](https://static.igem.org/mediawiki/2021/b/b1/T--Moscow_City--Troubleshooting_on_Protocols_%282%29.pdf)

Test

gRNA and the target site of the viral RNA we will buy ready-made because this way there is a guarantee that we will definitely get the RNA in a short time. For the further realization of the test, we will get EHV-1 and EHV-4 samples from our European colleagues.

In addition, we need to consider the need to use the control at each step of the test, to which we will not add viral RNA. This is due to the following: if a lot of chimeric protein: then the inteins will combine earlier, even though trans-splicing is a low-efficiency process. And in vitro detection of dcas13a target RNA is a fast-going reaction. Thus, the ribonucleoprotein complex quickly finds the RNA in the right place and sits on it. The beta-lactamase fragments converge with the inteins, and trans-splicing occurs because of the spatial proximity of the intein fragments (in the case of the non-intein system, the beta-lactamase fragments converge and interact).

**We will check the work of our detection system in stages:**
First, we need to assemble our system from all the components we have. To do this, we first place separately in 4 test tubes in a special buffer different chimeric proteins and the gRNA corresponding to each one. After a minute, these solutions can already be used, since the dCas13a protein is joined to the gRNA in almost two minutes.

Mix in pairs the solutions of the intein system and separately the non-intein system. Then we quickly add viral RNA to the protein solutions - to all but the control. Then we add 3-5 drops of 0.5 mg/ml Nitrocefin to 1 ml of broth suspension. The appearance of red color within 20-30 min. Indicates beta-lactamase activity.

All concentrations of the components must be chosen experimentally. However, we can rely on our experience with similar systems and take preliminary values of the component concentrations from the articles:

* 3 μL 1000nM gRNA (100nM final) and 2 μL 0.25uM fusion protein into 8.5 uL of 1X HEPES buffer (20mM HEPES, 150mM KCl, pH 7.5), and incubated at 25℃ for 10 mins.
* Add 3μL 30nM target (3 nM final) to the mixture of two protein-gRNA complexes and incubate at 37°C for 30 minutes.
* Pipet samples (30uL per well) into a 96-well black-welled plate (warmed to 37°C)

In our case, 37°C is the optimal temperature for dcas13a and for protein trans splicing, which is important for the intein system.

![](https://static.igem.org/mediawiki/2021/4/4f/T--Moscow_City--tild3965-6633-4466-a262-353033636238_-_empty_5_-_untitled_slide_1.png)

Results

Targeted viral RNA sites were selected, through binding to which our paired dCas13 system would provide specific detection of EHV-1 and EHV-4 viruses.

Molecular dynamics of the beta-lactamase cleaved into two fragments were performed with a trajectory length of 20 ns, from which the free fragment binding energy was calculated by the MM-PBSA method. The energy was negative, indicating that the formation of their complex is energetically advantageous.

We have determined the most likely mutual arrangement and their conformation relative to each other. The minimum energy shows that this is the state most 'convenient' for proteins. We can see with high probability this very complex, but its further behaviour in the buffer will only be shown by dynamics. It will also show binding energy. Binding energies and pathway analysis can already point to the biological meaning and biological effect.

We obtained one component of the system: it is dCas13-C\_lact-int - it successfully passed the screening stage. But its obtaining caused us some difficulties due to the inefficiency of ligation. To overcome this, instead of the 1h elongation suggested in the protocol, we performed it for 12h and the result was positive. We will take this into account in the future.

At the same time, we developed a detailed plan for performing experiments in the laboratory to implement our idea

**Reference**
Myhrvold, C. et al. Field-deployable viral diagnostics using CRISPR-Cas13. Science 360, 444–448 (2018).

Garcia-Venzor, Alfredo et al. “SARS-CoV-2 Direct Detection Without RNA Isolation With Loop-Mediated Isothermal Amplification (LAMP) and CRISPR-Cas12.” Frontiers in medicine vol. 8 627679. 17 Feb. 2021,

Gadkar, V.J., Goldfarb, D.M., Gantt, S. et al. Real-time Detection and Monitoring of Loop Mediated Amplification (LAMP) Reaction Using Self-quenching and De-quenching

Fluorogenic Probes. Sci Rep 8, 5548 (2018).

Lubis et al. (2016) Trends in Food Science 7 Technology. 58:55

Paulus, H. (2000). Protein Splicing and Related Forms of Protein Autoprocessing. Annual Review of Biochemistry, 69(1), 447–496.

S V Alexeyenkova and K P Yurov 2020 IOP Conf. Ser.: Earth Environ. Sci. 548 042041
https://www.biovision.com/beta-lactamase-activity-colorimetric-assay-kit.html

Brezgin S, Kostyusheva A, Kostyushev D, Chulanov V. Dead Cas Systems: Types, Principles, and Applications. International Journal of Molecular Sciences. 2019; 20(23):6041.

Ooi, K.H., Liu, M.M., Tay, J.W.D. et al. An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing. Nat Commun 12, 1739 (2021).

Myhrvold, Cameron et al. “Field-deployable viral diagnostics using CRISPR-Cas13.” Science (New York, N.Y.) vol. 360,6387 (2018): 444-448.

Huang et al. (2020). RT‐lamp for rapid diagnosis of coronavirus SARS‐COV‐2. Microbial Biotechnology, 13(4), 950–961.

Jung, J.K., Alam, K.K., Verosloff, M.S. et al. Cell-free biosensors for rapid detection of water contaminants. Nat Biotechnol 38, 1451–1459 (2020).

Zhang Y, Qian L, Wei W, Wang Y et al. Paired Design of dCas9 as a Systematic Platform for the Detection of Featured Nucleic Acid Sequences in Pathogenic Strains. ACS Synth Biol. 2017 Feb 17;6(2):211-216.

https://research.fredhutch.org/content/dam/stripe/hahn/methods/biochem/pet.pdf

[![Project](https://static.igem.org/mediawiki/2021/6/6b/T--Moscow_City--tild3331-3761-4334-a235-353066306137_12.svg)](https://2021.igem.org/Team%3AMoscow_City)

© 2021 iGEM Moscow City

* [Home page](https://2021.igem.org/Team%3AMoscow_City)
* [Description](https://2021.igem.org/Team%3AMoscow_City/Description)
* [EHV](https://2021.igem.org/Team%3AMoscow_City/EHV)
* [Contribution](https://2021.igem.org/Team%3AMoscow_City/Contribution)

* [Human Practices](https://2021.igem.org/Team%3AMoscow_City/Human_Practices)
* [Collaborations](https://2021.igem.org/Team%3AMoscow_City/Collaborations)
* [Implementation](https://2021.igem.org/Team%3AMoscow_City/Implementation)
* [Communication](https://2021.igem.org/Team%3AMoscow_City/Communication)

* [Engineering](https://2021.igem.org/Team%3AMoscow_City/Engineering)
* [Lab Book](https://2021.igem.org/Team%3AMoscow_City/Lab_book)
* [Model](https://2021.igem.org/Team%3AMoscow_City/Model)
* [Attributions](https://2021.igem.org/Team%3AMoscow_City/Attributions)

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# Team:Moscow City/Experiments

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# 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

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Moscow City/Results

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# 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.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)