iGEM 2021 — Team Moscow

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

# Engineering Success

## Introduction

Constant search for ideas, design, and attempts to improve the system, as well as detailed planning of all the stages of experiments represent the engineering success of our team. Unfortunately, we were not able to carry out many experiments in the laboratory due to limitations, so the focus of our work shifted to the dry laboratory.

Here are some detailed examples of how we went through the engineering cycle, what difficulties we faced and how we overcame them.

### Imagine

At first, we thought about creating an objective test for determining depressive disorder. We wanted to find a biomarker that could accurately identify depression and would not overlap with other mental diseases. MicroRNAs turned out to be such a marker. After conducting an extensive meta-analysis ( [read more](https://2021.igem.org/Team%3AMoscow/miRNA_analysis) here) and making sure that such a test would be relevant, we began to look for a microRNAs detecting method.

![](https://static.igem.org/mediawiki/2021/a/a5/T--Moscow--EngSuccess1.png)

### Research and general design

The existing main method for assessing the microRNA qPCR expression is not perfect, although it is sensitive. We chose the system with dCas9 and split GFP since a similar system with horseradish peroxidase (HRP) was promising high sensitivity, specificity, and low costs. Moreover, the use of a fluorescent protein would make it possible to multiplex the system in the future.

## Engineering design cycle: dumbbell probe

### Design

To increase the chances of GFP fragments interacting with each other, we decided to try attaching two dCas9 in different places with different guide RNAs so that the N and C ends would interact precisely and increase the likelihood of GFP maturation in the entire synthesized sample. In the first variant of the system, the two dCas9 proteins are attached to a stem region of the dumbbell probe as shown in the figure below.

![]( https://static.igem.org/mediawiki/2021/7/7d/T--Moscow--EngSuccess2.png)

### Build

You can read more about how the dumbbell probe was constructed on the  [dry lab page](https://2021.igem.org/Team%3AMoscow/Model) . Shortly, we chose the length of different nucleotide regions, the landing site of the Cas proteins, the landing site of miRNA, and the breakpoint. All design principles were compiled with literature analysis, as well as consultation with experts.

### Test and Learn

The obtained sequences are shown in the following figures, as well as the visualization obtained with RNAfold program[1] using secondary structure modeling.

![]( https://static.igem.org/mediawiki/2021/e/e3/T--Moscow--EngSuccess3.png)
![]( https://static.igem.org/mediawiki/2021/d/dd/T--Moscow--EngSuccess4.png)

However, we found two flaws in the received dumbbell probe:

1. The double-stranded part is too long (about 90 nucleotides), and it lowers the chances of amplification- Sample without microRNA and subsequent amplification produces background noise

### Design

We decided to redo the design of the dumbbell probe. We put one dCas9 protein on a double-stranded part, and the other on a single-stranded part of the probe.

![](https://static.igem.org/mediawiki/2021/5/53/T--Moscow--EngSuccess5.png)

As a result, the length of the double-stranded part was 31 nucleotides, and the background signal was eliminated since the second Cas protein is attached only to the complementary sequence of the single-stranded part.

You can check more details in the dry lab section.

## Engineering design cycle: dCas9 cloning

### Design

For the assembly of the resulting plasmid containing fused dCas9 split GFP, we took into account some of the problems the team Moscow had faced in 2019. Due to poor solubility, dCas9 proteins fused with N- and C-ends of β-lactamase were poorly purified; during work after iGEM, it was shown that adding MBP protein to the His-tag and an additional purification step solves this problem. Therefore, we wanted to linearize plasmid pMJ841 and insert split GFP with new linkers.

Primer design is a non-trivial, everyday laboratory task. So we thought. But the dark and full of horrors world of scientific life hid a couple of surprises for us.

At first, we just picked up the primers with the help of Snapgene.

![](https://static.igem.org/mediawiki/2021/4/48/T--Moscow--EngSuccessT1.png)

### Build and Test

We have started to generate our DNA fragments using PCR. Some experiments were successful (green), but there we had faced difficulties. Some primers didn’t work (red)!

Snapgene.

![](https://static.igem.org/mediawiki/2021/c/ca/T--Moscow--EngSuccess6n.png)
![](https://static.igem.org/mediawiki/2021/9/90/T--Moscow--EngSuccess7n.png)
![](https://static.igem.org/mediawiki/2021/9/9f/T--Moscow--EngSuccess8n.png)
![](https://static.igem.org/mediawiki/2021/f/f6/T--Moscow--EngSuccess9n.png)
![](https://static.igem.org/mediawiki/2021/1/14/T--Moscow--EngSuccess11n.png)
![](https://static.igem.org/mediawiki/2021/e/e1/T--Moscow--EngSuccess12n.png)

### Learn

We decided to figure out where our mistake was and conducted a deep literary analysis.

1. First, we found that the formula for calculating Tm that SnapGene uses is not optimal, since it does not take into account the concentration of the salts we need, the added nucleotides, etc. SnapGene assumes a Na+ concentration of 50 mM, and we use Q5 polymerase[2] which buffer contains no Na, but a 2 mM Mg++.- The Q5 polymerase we use works at higher temperatures than the rest, and the manufacturer is in the protocol recommends using a special calculator https://tmcalculator.neb.com/#!/main of Tm- When choosing primers, it is necessary to check them for the formation of hairpins. The Tm for the strongest hairpin should be at least 50°C below the annealing temperature [3]. To check for them, you can use, for example, unafold [4].- Both self- and hetero-dimers should be reviewed. In general, the ΔG value for dimer analysis should be between 0 to −9 kcal/mole for the optimal design [5]- GC composition must be specific, for our protocol, it is 40-60%, differences in temperature between primers do not exceed 3-5 degrees.- The online analyzer[6] and a training video[7] for it helped us a lot, which turned out to be a lot of useful information.- Because we use an overlap PCR, linkers are sewn to our primers. And, as it turned out during verification, they make a significant contribution to the formation of homo and heterodimers.- Also worth considering that our linkers are long and rich for GC, so we have to add a Tm lowering agent to our reaction.- We have developed a design for a new batch of primers, in which we thoroughly tested everything with the help of [6] , and also tested and adapted the linkers.

### Design

After the failure with linearization, we decided to change the type of assembly and firstly get DNA constructs for fused dCas9-linker-NGFP and СGFP-linker-dCas9 by overhang PCR.

![](https://static.igem.org/mediawiki/2021/e/e3/T--Moscow--EngSuccess16n.png)

And then insert them into the linearized backbone containing the promoter, RBS, His-tag, MBP, and terminator.

### Build and Test

![](https://static.igem.org/mediawiki/2021/7/77/T--Moscow--EngSuccess13n.png)

Unfortunately, we had a very limited time to access the laboratory - 1 month. We did not conduct many experiments and faced the difficulties described above. However, a lot of theoretical preparation has been done that will help us achieve a real result in the future while going through the engineering cycle in different stages.

## Chapter 1: dCas9-splitGFP + sgRNA

### Cloning

As linearization did not go well we have changed the PCR design as described above. New primers provide the assembly of ORFs for NGFP-linker-dCas9 and dCas9-linker-CGFP constructs by overhang PCR (protocol for PCR Using Q5 DNA-polymerase is described here [8]. Primers were selected based on the optimal linker length obtained by modeling ( [Link to dry lab](https://2021.igem.org/Team%3AMoscow/Model)). PCR products will be phosphorylated (Protoco for phosphorylation with T4 PNK[9]) and blunt-end ligated (Ligation Protocol with T4 DNA Ligase[10]) to the linearized pMJ 841 (without dCas9 gene). At each step, we will analyze PCR products by agarose gel electrophoresis and clean them. ДВЕ КАРТИНКИ

After successful cloning, transformation, and sequence screening, we would carry out mutagenesis to remove illegal restriction sites. After all measurements of our test system in pEC-K-MBP backbone, we would transfer our transcription units in the iGEM pSB1C3 backbone with a Type IIS assembly method.

### Troubleshooting

We will use NEB guides to troubleshoot PCR [11] [12].

### Сompetent E. coli. preparation

We planned to use the E. coli Dh5α strain as a cloning host and strains BL21, Rosetta-gami 2 (DE3) pLysS for protein expression. Competent cells were prepared as described in the brief protocol below. To determine any inducer-dependent effects in E. coli BL21, Rosetta-gami 2 (DE3) pLysS the growth kinetics will be studied, both in the presence and absence of IPTG. To confirm the competence, the cells would be transformed with a control plasmid (2pg, 20pg, 200pg, 2 ng, 20 ng) and plated on an LB+selective antibiotic plate as a positive control.

### Protocol:

1. Streak out frozen glycerol stock of bacterial cells (-80°C) onto LB plate. Grow overnight 37°C.- Transfer several colonies into 4 ml LB and grow at 18°C with continuous stirring (230 rpm) until reaching OD600 = 0.4- After transfer cells into 500 ml of medium and continue cultivation.

All next steps should be carried out in the cold.

1. Pellet cells down at 4000 rpm, 4°C for 10 minutes, discard the supernatant and resuspend pellet in 50 ml of cooled Inoue Buffer (MnCl2 4H2O 55 mM, CaCl2 2H2O 15 mM, KCl 250 mM, PIPES pH 6.7 10 mM)- Repeat 4th step- Add 4 ml of Inoue Buffer and 300 μl of DMSO to the pellet and incubate on ice for 10 minutes.- Aliquot cells in sterile tubes, freeze in liquid nitrogen, store at - 80°C.

### Troubleshooting

Check all chemicals and remake buffers. Make sure to use appropriate antibiotic concentrations.

### Transformation and Screening

Ligated products will be transformed into E.coli Dh5α host strain

### Brief protocol:

1. Thaw competent cells on ice for 10 minutes- Add ~ 100 ng of plasmid DNA, thoroughly resuspend, and incubate on ice for 15 minutes.- Heat shock at 42°C for 60 seconds.- Incubate on ice for 2 minutes and add 200 μl LB without antibiotics.- Cultivate cells for 45 minutes at 37°C with constant stirring.- Spread cells on LB+selective antibiotic(s) plate and incubate at 37°C overnight.

After that, we will select individual colonies, transfer each to a replica plate and perform colony PCR (Insert Screening Protocol described here https://international.neb.com/protocols/2013/12/27/insert-screening-protocols-e1202 ) using Orientation-Specific primers. PCR products will be analyzed by agarose gel electrophoresis. Plasmids with rightly oriented inserts will be extracted. Inserts then will be sequenced.

### Troubleshooting

If any troubles occur we will use this guide [13].

### Protein expression

We will use two E. coli expression strains: BL21 and Rosetta-gami 2(DE3)pLysS. We will transform expression plasmids into strains using the same protocol described in the transformation section.
The expression will be induced in the presence of IPTG (protocol is described here [14]). We will vary IPTG concentration (0.1, 0.5, 1.0 mM) and harvest 1 mL of cells from each culture (Induced and control) at the following time intervals: 0 hr, 1 hr, 2hr, 3hr, 4hr, 5hr. This is needed to finetune our protocols and choose a better strain. We will check the expression of our proteins (SDS-PAGE) as described here [14], [15].

### Protein purification

To purify our proteins we will use metal affinity enrichment followed by cation exchange chromatography. Before performing cation exchange chromatography MBP protein and His-tag will be separated from dCas9-GFP by TEV-protease following this protocol[16]. The full purification protocol is described here [17].

### Troubleshooting

We will use guides to troubleshoot metal affinity chromatography [18] and ion-exchange chromatography [19]

### sgRNA

sgRNAs will be generated using the Part:BBa\_K1689000. We will clone our target sequences into the generator. Complete sgRNAs will be generated by in vitro transcription using the T7 Invitrogen kit, following manufacturer instructions described here [20]. Products will be analyzed by 2% agarose gel electrophoresis. RNA will be cleaned up on GeneJET RNA columns and concentrations will be analyzed using Qubit RNA HS Assay Kit.

To determine the required concentrations for dCac9:sgRNA complex formation, we will conduct EMSA in 5% PAGE.

## Chapter 2: RCA

### Dumbbell probe preparation

Designed probes ( [link to dry lab](https://2021.igem.org/Team%3AMoscow/Model)) will be purchased as self-annealing precursor ssDNA oligonucleotides. Diluted oligonucleotides will be linearized to improve the phosphorylation efficiency (5 minutes heating at 95°C immediately followed by 5 minutes incubation on ice). 5’ 5-phosphorylation will be carried on the oligonucleotides using T4 PNK according to Protocol [21](except inactivation stage) and followed by annealing by slow cooling from 95°C to 4°C. Ligation will be performed using T4 DNA ligase.

### Brief protocol:

1. Mix on ice annealed 5-phosphorylated DNA (3.2 μM), T4 DNA ligase, ATP (1 mM), and T4 DNA ligase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM ATP, 10 mM DTT).- Incubate at 16°C for 16 hours- Inactivate ligase at 75°C for 20 min.

To digest all unligated ssDNA and dsDNA Exonuclease I and Exonuclease III will be added. The solutions will be incubated at 37C for 4 hours, followed by enzyme inactivation at 85°C for 20 minutes and then stepwise annealing from 85°C to 4°C to generate the desired secondary structure. To check the efficiency of ligation and digestion probes will be analyzed in 12% denaturing PAGE using the protocol described here [22].

### Troubleshooting

Increase ligation time and amount of ligase, similar to exonucleases.

### Rolling Circle Amplification

In this part we will verify that target microRNA can trigger RCA. Analysis of RCA performance will be monitored by real-time fluorescence measurement. This will help us to understand which probe is better to use in future experiments, adapt the RCA protocol, and find if our design has weak points.

### Brief protocol:

1. Mix dumbbell probe, miRNA, phi29 DNA polymerase reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT), dNTPs (1.0 mM), BSA (0.2 g/µL) 0.4XSYBR Green I, phi29 DNA polymerase (0.2 units/µL). Note: we will vary concentrations of dumbbell probe and miRNA- Conduct reaction on Real-Time PCR system in real time for 3h (1 min intervals).

To study the specificity of our system we will also conduct experiments with different DNA probes (various ligation sites and complementary to miRNA regions) and miRNAs (1, 2 or 3 mismatching nucleotides).

## Chapter 3: Proovement of conсept

Step 1. Perform RCA with dumbbell probe and target miRNA. We will adapt the protocol described above in the Rolling Circle Amplification section.

Step 2. Preincubate dCac9-splitGFP with sgRNA. It is important to separately incubate dCas9-NGFP with sgRNA which ought to bind to the hairpin's stem and dCas9-CGFP with sgRNA complementary to the ssDNA region. This step should let us reach the best achievable fluorescence intensity because in this position dCas9-splitGFP protein pairs most likely form mature GFP.

### Brief protocol:

1. Mix 10XCas9 Nuclease Reaction Buffer, sgRNA, Cas9-splitGFP, Nuclease-free water in a total volume of 20µL- Incubate for 15 min at 37°C.

Step 3. Add preincubated dCac9-splitGFP:sgRNA to RCA products. And wait for GFP recovery. We will measure fluorescence intensity at different times using Zeiss LSM 710 Confocal Microscope to determine optimal incubation time for our test system. We will also perform measurements with different microRNA concentrations to find the dynamic range and detection limit of our technique. Also, we will try to detect signal using our hardware.

## References:

1. http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi- https://international.neb.com/protocols/2013/12/13/pcr-using-q5-high-fidelity-dna-polymerase-m0491- https://www.idtdna.com/pages/support/faqs/what-secondary-structure-considerations-need-to-be-included-when-designing-primers-for-pcr-- http://www.unafold.org/mfold/applications/dna-folding-form.php- https://www.idtdna.com/pages/support/faqs/what-secondary-structure-considerations-need-to-be-included-when-designing-primers-for-pcr-- https://www.idtdna.com/calc/analyzer- https://www.youtube.com/watch?v=hB8YSgE97H0- https://international.neb.com/protocols/2013/12/13/pcr-using-q5-high-fidelity-dna-polymerase-m0491- https://international.neb.com/protocols/0001/01/01/non-radioactive-phosphorylation-with-t4-pnk-or-pnk3-phosphatase-minus- https://international.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202- https://international.neb.com/tools-and-resources/troubleshooting-guides/pcr-troubleshooting-guide and ligation- https://international.neb.com/tools-and-resources/troubleshooting-guides/troubleshooting-guide-for-ligases- https://international.neb.com/tools-and-resources/troubleshooting-guides/troubleshooting-guide-for-cloning- https://www.biologicscorp.com/blog/iptg-induction-protocol/#.YXISOxozaMp- http://microbiology.ucdavis.edu/privalsky/sites/default/files/SDS-PAGE\_0.pdf- https://www.urmc.rochester.edu/MediaLibraries/URMCMedia/labs/wedekind-lab/documents/TEV-Protease-Cleavage-Protocol.pdf- https://www.mdpi.com/2409-9279/1/2/17/html- https://www.sigmaaldrich.com/RU/en/technical-documents/technical-article/protein-biology/protein-purification/troubleshooting1- https://www.sigmaaldrich.com/RU/en/technical-documents/technical-article/protein-biology/protein-purification/ion-exchange-chromatography-troubleshooting- https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2FMAN0012652\_TranscriptAid\_T7\_High\_Yield\_Transcription\_UG.pdf- https://international.neb.com/protocols/0001/01/01/non-radioactive-phosphorylation-with-t4-pnk-or-pnk3-phosphatase-minus- http://microbiology.ucdavis.edu/heyer/wordpress/wp-content/uploads/2013/11/DNA\_PAGE.pdf- https://www.idtdna.com/pages/support/faqs/what-secondary-structure-considerations-need-to-be-included-when-designing-primers-for-pcr-

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

* [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/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.

* [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)