iGEM 2021 — Team MIPT_MSU

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# Team:MIPT MSU/Notebook

[ ]

![Logo](https://static.igem.org/mediawiki/2021/a/a5/T--MIPT_MSU--logo-round-basic.PNG)

* Project
* [Team](https://2021.igem.org/Team%3AExample2/Team)
* [Parts](https://2021.igem.org/Team%3AExample2/Parts)
* Human Practice
* [Judging Form](https://igem.org/2021_Judging_Form?id=3942)

# 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:MIPT MSU/Engineering

[![](https://static.igem.org/mediawiki/2021/c/c3/T--MIPT_MSU--logo_header.png)](https://2021.igem.org/Team%3AMIPT_MSU)

[project](https://2021.igem.org/Team%3AMIPT_MSU/Description)
![](https://static.igem.org/mediawiki/2021/d/d8/T--MIPT_MSU--aa-arrow.svg)

[Description](https://2021.igem.org/Team%3AMIPT_MSU/Description)[Engineering
success](https://2021.igem.org/Team%3AMIPT_MSU/Engineering)[Model](https://2021.igem.org/Team%3AMIPT_MSU/Model)[Proof of
concept](https://2021.igem.org/Team%3AMIPT_MSU/Proof_Of_Concept)[Implementation](https://2021.igem.org/Team%3AMIPT_MSU/Implementation)

[team](https://2021.igem.org/Team%3AMIPT_MSU/Team)
![](https://static.igem.org/mediawiki/2021/d/d8/T--MIPT_MSU--aa-arrow.svg)

[Team
members](https://2021.igem.org/Team%3AMIPT_MSU/Team)[Attributions](https://2021.igem.org/Team%3AMIPT_MSU/Attributions)[Collaboration](https://2021.igem.org/Team%3AMIPT_MSU/Collaborations)

[human
practices](https://2021.igem.org/Team%3AMIPT_MSU/Human_Practices)
![](https://static.igem.org/mediawiki/2021/d/d8/T--MIPT_MSU--aa-arrow.svg)

[Human
practices](https://2021.igem.org/Team%3AMIPT_MSU/Human_Practices)[Education](https://2021.igem.org/Team%3AMIPT_MSU/Communication)[Excellence
in another area](https://2021.igem.org/Team%3AMIPT_MSU/Excellence_In_Another_Area)[Best
Education](https://2021.igem.org/Team%3AMIPT_MSU/Education)

[contribution](https://2021.igem.org/Team%3AMIPT_MSU/Contribution)
![](https://static.igem.org/mediawiki/2021/d/d8/T--MIPT_MSU--aa-arrow.svg)

[Parts](https://2021.igem.org/Team%3AMIPT_MSU/Parts)[contribution](https://2021.igem.org/Team%3AMIPT_MSU/Contribution)

[lab
work](https://2021.igem.org/Team%3AMIPT_MSU/Engineering)
![](https://static.igem.org/mediawiki/2021/d/d8/T--MIPT_MSU--aa-arrow.svg)

[Experiments and
protocols](https://2021.igem.org/Team%3AMIPT_MSU/Experiments)[results](https://2021.igem.org/Team%3AMIPT_MSU/Results)

Engineering success

![](https://static.igem.org/mediawiki/2021/6/6e/T--MIPT_MSU--new_eng_top.svg)

![](https://static.igem.org/mediawiki/2021/f/ff/T--MIPT_MSU--Design_1.svg)

We aim to develop a precise drug delivery system to target cells. After searching through the
literature,
we selected vesicles as a base for our system. Their established role in cell to cell communications,
naturally low toxicity
and immunogenicity, is their strong advantage . A point to note, this system needs targeting and packaging
cargo.
The first one is the syncytin that specifically binds to receptors on the surface of certain cancer cells
(e.g. colorectal adenocarcinoma). In this way, the vesicles fuse with the target cell membrane and bypass
the endosomal pathway.
The vesicular cargo is mRNA modified with a psi RNA packaging signal that can be recognized by the RNA
binding domain.
To see more of our delivery system background, go to our vesicles project [Description](https://2021.igem.org/Team%3AMIPT_MSU/Description).

![](https://static.igem.org/mediawiki/2021/d/d9/T--MIPT_MSU--build_1.svg)

Cloning I (Primary structures)

The first part of our plan consisted in cloning the endogenous retroviral protein syncytin
(ERVW-1) into an Evrogen turboGFP N vector for amplification in E. coli. This
vector was chosen
for studying how fusion between Syncytin and GFP would affect its activity.
Therefore, we cloned two versions of syncytin, first with stop codon and second one without it,
into the same vector to study the potential efficiency differences. Syncytin was initially
obtained by amplifying it from the MCF7’s cDNA. We isolated total RNA from MCF-7 cells and performed
reverse transcription followed by PCR for the ERVW-1 gene. Using electrophoresis,
we validated the result of obtaining cDNA of the ERVW-1 gene from MCF-7 cells and three fragments
of different lengths were found, one of which resembled the expected length of ERVW-1.
We assumed that this is due to the fact that our primers also annealed some other sequences present
in the MCF-7 culture, which were also present in the total cDNA. In this regard,
it was decided to cut a band corresponding to the length of ERVW-1 from the gel and repeat PCR.
However, in the next round of PCR, we also observed three bands corresponding to the same lengths.
We believe that this is due to poor separation of these three fragments on the gel, due to which some
part of the non-target two genes nevertheless, when excised from the gel, got into the excised piece of gel
and was re-amplified.
After cloning the sample obtained from MCF7 and sending it for sequencing in Evrogen,
we discovered that the gene was highly mutated. Because of this failure, we decided to order the synthesis
of the syncytin gene from Twist Bioscience. Having obtained this gene, we repeated PCR and got the required
fragment.

Figure 1. Blast Alignment between sequenced fragment of Syncytin obtained
from MCF7 cell line (top)
and expected Syncytin sequence (bottom). Total of 51 mutations were found only in this region, including
insertions and deletions.
Because of a high percentage of errors, we decide to use the services of Twist Bioscience and ordered gene
synthesis.

![](https://static.igem.org/mediawiki/2021/3/30/T--MIPT_MSU--syncytin_mutated_1.png)

For the second cloning, we used the b-galactosidase (lacZ) gene, from the iGEM kit obtained by
PCR
and cloned it into the Evrogen turboGFP C vector. This combination of reporters makes it possible
to detect the GFP luminescence, and also to carry out blue-white selection. These reporters will
be later used to check that sorting and transporting vesicles’ content was successful. In addition,
this construction will be used as a backbone for fragments in the Clone II part.

Last cloning in this series was aimed to solve the RNA recognition problem. For it, we clone
gag-polyprotein,
which includes the RNA-binding domain (RBD) into the lentiviral vector, for stable transfection.
Gag protein will be responsible for recognising psi-RNA packaging signal and
formation of vesicles
through ESCRT machinery. The gag-polyprotein gene was kindly provided by our instructor Evgenii Lunev
(Institute of Gene Biology, Russian Academy of Sciences), and the plasmid was given by the Laboratory
of Cell Signalling Regulation. To see more about cloning I, go to our [Experiments
page](https://2021.igem.org/Team%3AMIPT_MSU/Experiments).

Cloning II (Gibson Assembly)

After consulting with the Marlin Biotech company, we listened to their
advice, and started this stage by optimizing
the efficiency of RNA translation. Adding IRES will help facilitate the entry of the ribosome onto our mRNA
and thus
increase the expression of the target mRNA protein. Additionally, we learned from Marlin that for successful
interaction with RBD, other 5'-UTR regions of HIV-1 (and not just the psi-short signal) may also be
required.
Therefore, we amplified psi-short and full 5'-UTR region (psi-long) RNA packaging signals. Psi-short was
obtained
from the Laboratory of Cell Signalling Regulation, while psi-long was obtained by gene synthesis from Evrogen.
As all basic components were obtained, we used Gibson Assembly to create these constructions:

5’ - psi(short) - IRES - GFP - lacZ -3’

5’ - psi(short) - GFP -lacZ - 3’

5’ - psi(long) - IRES -GFP - lacZ - 3’

5’ - psi(long) - GFP - lacZ -3’

5’ - IRES - GFP - lacZ - 3’

All construction except for the 3rd one were obtained on the first try.
This may be explained by the fact that 3rd construction demanded assembly of larger
fragments then any other. Therefore, we decided to double the added weight of fragments,
which resulted in successful assembly.

First four were used to test the effect of IRES/ Psi-short vs Psi-long,
and the last one is a negative control addition of which was also suggested by Marlin company.
To see more about cloning II, go to our [Experiments page](https://2021.igem.org/Team%3AMIPT_MSU/Experiments).

![](https://static.igem.org/mediawiki/2021/7/7d/T--MIPT_MSU--transfection_1.svg)

By using plasmids obtained in the previous steps, we transfected MCF-7 cells with them by
electroporation using the Lonze kit. Therefore, we had 5 wells with Syncytin (with stop codon),
gag-polyprotein and one of the constructions obtained in a previous step. Two days after nucleofection we
observed luminescence in all wells. However, those samples that had constructions with psi-long showed
significantly lower luminescence than the rest of them.

Obtaining Vesicles:

Vesicles are naturally expressed by the MCF7 cells in the supernatant. Therefore we immediately
collected and dripped it onto HT-29 cells. HT-29 was chosen as a target for our vesicles as they contain
syncytin-1 receptors. After a single day since adding vesicles, we observed GFP luminescence only in the
dish to which vesicles with 5’-psi(short)-IRES-GFP-lacZ-3’ construction were added.

![](https://static.igem.org/mediawiki/2021/e/e2/T--MIPT_MSU--test_1.svg)

After obtaining the plasmids we tested each of them by gel electrophoresis (Figures 2 to 4),
and by sending them for sequencing to Evrogen, to be sure that all data is correct. More on that at the
[Results page](https://2021.igem.org/Team%3AMIPT_MSU/Results).

The gel electrophoresis results after Syncytin amplification
from sequence synthesised by Twist Bioscience.
Sample 1 (Figure 2) - syncytin amplified by using primers with stop codon. Sample 2 (Figure 2) - syncytin amplified by using
primers without stop codon.
Left column presents a 1kb+ Marker from Evrogen.

![](https://static.igem.org/mediawiki/2021/b/b9/T--MIPT_MSU--1st_type_%28with%29_1.png)
![](https://static.igem.org/mediawiki/2021/c/cb/T--MIPT_MSU--1st_type_%28without%29_1.png)

![](https://static.igem.org/mediawiki/2021/f/f0/T--MIPT_MSU--3rd_type_1.png)
Figure 3. The gel electrophoresis results after gag amplification after
cloning
it into a lentiviral vector. Left column presents 1kb+ Marker from Evrogen.
Right column presents an amplified gag (weight 1503 bp).

Figure 4. The gel electrophoresis results of 5 construction made with
Gibson Assembly.
Left to right: 1kb+ Evrogen Marker, psi(short)-IRES-GFP-lacZ 1295 bp, psi(short)-GFP-lacZ 747 bp,
psi(long)-IRES-GFP-lacZ 1525bp, psi(long)-GFP-lacZ 973bp,IRES-lacZ 1149 bp. All constructions presented by
bright white bands.

![](https://static.igem.org/mediawiki/2021/1/17/T--MIPT_MSU--2nd_type_1.png)

When it comes to testing our system as a whole, addition of supernatant containing vesicles to
the HT-29 cells revealed that, vesicles containing 5’-psi(short)-IRES-GFP-lacZ-3’ structure are able to
cause GFP luminescence in the targeted cells (Figure 5). More details on the [Experiments page](https://2021.igem.org/Team%3AMIPT_MSU/Experiments)

Figure 5. HT-29 cells treated with vesicles with 5’-psi(short)-IRES-GFP-lacZ-3’
construction. Expression of GFP was observed after a single day since treatment.

![](https://static.igem.org/mediawiki/2021/d/d2/T--MIPT_MSU--Proof_of_concept_1.png)

![](https://static.igem.org/mediawiki/2021/a/a2/T--MIPT_MSU--learn_1.svg)

Development of a targeted vesicle based drug delivery system presented a complicated challenge.
Due to severe time constraints caused by the late and irregular provision of necessary laboratory equipment,
the fact that gene synthesis provided by the iGEM is unavailable in Russia, and that use of the primary cell
line is a poor source of unmutated DNA, we were unable to complete the latest steps of our project. However,
even with incomplete data we still manage to find useful insights. First is that you should be very cautious
during amplification of long gene sequences from cDNA obtained from primary cell lines. Using MCF7 as a source
of the ERVW-1 gene proved to be unreliable, revealing multiple mutations and 2 off target products.
Furthermore,
cloning problems continued during implementation of Gibson assembly, as it was necessary to connect two
rather
large fragments: psi-long (356 nt) and IRES (551 nt). This problem was solved by adding doubled amounts of
both
psi-long and IRES to the reaction mixture. For the further step, from the Pavel
Makarevich we learned that,
initially proposed for vesicle formation, hTERT cell line is poorly transfected, however, they are highly
susceptible to transduction. Therefore, we decided to change the producing cell line to MCF7.

Which showed appropriate results after nucleofection, as GFP luminescence was observed in all
wells. Moreover, MCF7 cells expressed enough cells into supernatant that even without advance filtering or
vesicle separation, our vesicles containing 5’-psi(short)-IRES-GFP-lacZ-3’ construction were able to cause
GFP luminescence within target cells. Other constructions did not show the similar results, suggesting that
presence of IRES does assist the translation of GFP mRNA, and that vesicles containing construction with
psi-long require longer time to be produced in meaningful quantities (more on that on the [Proof Of Concept page](https://2021.igem.org/Team%3AMIPT_MSU/Proof_Of_Concept)). All
together we believe that even without finishing all the planned steps we proved that our system is working,
and we will carry on our experiments to lay a foundation for future potential systems. Which with proper
care could be used as potential therapeutic treatment for numerous diseases.
**And hopefully deliver a cancer
free world to you.**

[![](https://static.igem.org/mediawiki/2021/c/c9/T--MIPT_MSU--arrow_top.svg)](#top)

[Design](#Design)

[Build](#Build)

[Transfection](#Transfection)

[Test](#Test)

[Learn](#Learn)

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

# Team:MIPT MSU/Experiments

[![](https://static.igem.org/mediawiki/2021/c/c3/T--MIPT_MSU--logo_header.png)](https://2021.igem.org/Team%3AMIPT_MSU)

[project](https://2021.igem.org/Team%3AMIPT_MSU/Description)
![](https://static.igem.org/mediawiki/2021/d/d8/T--MIPT_MSU--aa-arrow.svg)

[Description](https://2021.igem.org/Team%3AMIPT_MSU/Description)[Engineering
success](https://2021.igem.org/Team%3AMIPT_MSU/Engineering)[Model](https://2021.igem.org/Team%3AMIPT_MSU/Model)[Proof of
concept](https://2021.igem.org/Team%3AMIPT_MSU/Proof_Of_Concept)[Implementation](https://2021.igem.org/Team%3AMIPT_MSU/Implementation)

[team](https://2021.igem.org/Team%3AMIPT_MSU/Team)
![](https://static.igem.org/mediawiki/2021/d/d8/T--MIPT_MSU--aa-arrow.svg)

[Team
members](https://2021.igem.org/Team%3AMIPT_MSU/Team)[Attributions](https://2021.igem.org/Team%3AMIPT_MSU/Attributions)[Collaboration](https://2021.igem.org/Team%3AMIPT_MSU/Collaborations)

[human
practices](https://2021.igem.org/Team%3AMIPT_MSU/Human_Practices)
![](https://static.igem.org/mediawiki/2021/d/d8/T--MIPT_MSU--aa-arrow.svg)

[Human
practices](https://2021.igem.org/Team%3AMIPT_MSU/Human_Practices)[Education](https://2021.igem.org/Team%3AMIPT_MSU/Communication)[Excellence
in another area](https://2021.igem.org/Team%3AMIPT_MSU/Excellence_In_Another_Area)[Best
Education](https://2021.igem.org/Team%3AMIPT_MSU/Education)

[contribution](https://2021.igem.org/Team%3AMIPT_MSU/Contribution)
![](https://static.igem.org/mediawiki/2021/d/d8/T--MIPT_MSU--aa-arrow.svg)

[Parts](https://2021.igem.org/Team%3AMIPT_MSU/Parts)[contribution](https://2021.igem.org/Team%3AMIPT_MSU/Contribution)

[lab
work](https://2021.igem.org/Team%3AMIPT_MSU/Engineering)
![](https://static.igem.org/mediawiki/2021/d/d8/T--MIPT_MSU--aa-arrow.svg)

[Experiments and
protocols](https://2021.igem.org/Team%3AMIPT_MSU/Experiments)[results](https://2021.igem.org/Team%3AMIPT_MSU/Results)

Experiments

![](https://static.igem.org/mediawiki/2021/4/43/T--MIPT_MSU--SynCytin_Cloning.svg)

This protocol shows how we cloned Syncytin into Evrogen turboGFP N vector.

**Materials:**

1. cDNA from MCF7 cell line or order Gene synthesis.
2. Primers:

   F-syncytin - GGAAAGAATTCATGGCCCTCCCTTATCATATTTTTCTCT

   R-s-stop - AACCGGTCTAACTGCTTCCTGCTGAATTGGG

   R-s - AACCGGTAAACTGCTTCCTGCTGAATTGGG

   F-CMV - ATAAGCAGAGCTGGTTTAGT

   R-GFP - AACTCCACGCCGTT
3. Taq, Q5 polymerase and buffers for them.
4. Evrogen turboGFP N vector plasmid.
5. Restriction: AgeI, EcoRI.
6. Thermo Fisher FastDigest x10 buffer.
7. T4 DNA ligase, 10x T4 ligation buffer.
8. Competent cells (XL10 Gold).

**Procedure:**

1. Syncytin PCR:

   | Reagent | Volume, μl | PCR program |
   | --- | --- | --- |
   | 2X MasterMix | 10 | 1. 98 C → 30'' 29X: |
   | F-syncytin | 1 | 2. 98 C → 15'' |
   | R-s-stop/s | 1 | 3. 60 C → 30'' |
   | DNA | 0.5 | 4. 72 C → 1' |
   | H2O | 7.5 | 5. 72 C → 1' |
   | **Total** | 20 | 6. 4 C → inf |
2. Gel electrophoresis, and use kit to purify DNA from gel.
3. Restriction:

   | Reagent | Volume, μl | Restriction condition |
   | --- | --- | --- |
   | FD 10x green buffer | 1.5 | 2 hours at 37 С |
   | AgeI | 0.5 |  |
   | EcoRI | 0.5 |  |
   | Syncytin | 10 |  |
   | H2O | 2.5 |  |
   | **Total** | 15 |  |

   | Reagent | Volume, μl | Restriction condition |
   | --- | --- | --- |
   | FD 10x green buffer | 1.5 | 2 hours at 37 С |
   | AgeI | 0.5 |  |
   | EcoRI | 0.5 |  |
   | Syncytin | 0.5 |  |
   | H2O | 12 |  |
   | **Total** | 15 |  |
4. Purify digested DNA from mixture.
5. Ligation:

   | Reagent | Volume, μl | Ligation condition |
   | --- | --- | --- |
   | 10x T4 liogation buffer | 1.5 | Overnight at 16 C |
   | T4 ligase | 0.5 |  |
   | Syncytin | 10 |  |
   | TurboGFP N | 3 |  |
   | H2O | 0 |  |
   | **Total** | 15 |  |
6. Transformation (read more here)
7. Screening colonies:

   | Reagent | Volume, μl | PCR program |
   | --- | --- | --- |
   | 5x screen mix | 3 | 1. 95 C → 3' 29X: |
   | F | 0.5 | 2. 95 C → 30'' |
   | R | 0.5 | 3. 54 C → 30'' |
   | RNAse | 0.1 | 4. 72 C → 1:45'' |
   | H2O | 11 | 5. 72 C → 5' |
   | **Total** | 15 | 6. 12 C → inf |
   | **DNA** | 0 |  |
8. Growth positively screened colonies in LB overnight.
9. Use Miniprep to obtain plasmid DNA.
10. Confirm obtained DNA by using PCR:

    | Reagent | Volume, μl | PCR program |
    | --- | --- | --- |
    | 5x screen mix | 3 | 1. 95 C → 3' 29X: |
    | F | 0.5 | 2. 95 C → 30'' |
    | R | 0.5 | 3. 54 C → 30'' |
    | H2O | 10.5 | 4. 72 C → 1:45'' |
    | **DNA** | 0.5 | 5. 72 C → 5' |
    | **Total** | 15 | 6. 12 C → inf |

![](https://static.igem.org/mediawiki/2021/1/1a/T--MIPT_MSU--Lacz_Cloning.svg)

This protocol shows how we cloned LacZ into Evrogen turboGFP C vector.

**Materials:**

1. LacZ plasmid from iGEM kit transformed into DH5alpha cells.
2. Primers:

   F-lacZ - ATGCCTCGAGCAATGACCATGATTACGGATTCACTGGC

   R-lacZ - ATGCGGTACCCATTATTTTTGACACCAGACCAACTGG
3. Evrogen Taq, NEB Q5 polymerase and buffers for them.
4. Evrogen turboGFP C vector plasmid.
5. Restrictions: XhoI, KpnI.
6. Thermo Fisher FastDigest x10 buffer.
7. T4 DNA ligase, 10x T4 ligation buffer.
8. Competent cells (XL10 Gold).

**Procedure:**

1. LacZ PCR:

   | Reagent | Volume, μl | PCR program |
   | --- | --- | --- |
   | 2X MasterMix | 10 | 1. 98 C → 30'' 29X: |
   | F-lacZ | 1 | 2. 98 C → 15'' |
   | R-lacZ | 1 | 3. 60 C → 30'' |
   | **DNA** | 0.5 | 4. 72 C → 1' |
   | H2O | 7.5 | 5. 72 C → 1' |
   | **Total** | 20 | 6. 4 C → inf |
2. Gel electrophoresis, and use Evrogen or Thermo Fisher kit to purify DNA from gel
3. Restriction:

   | Reagent | Volume, μl | Restriction condition |
   | --- | --- | --- |
   | FD 10x green buffer | 1.5 | 2 hours at 37 С |
   | KpnI | 0.5 |  |
   | XhoI | 0.5 |  |
   | LacZ | 10 |  |
   | H2O | 2.5 |  |
   | **Total** | 15 |  |

   | Reagent | Volume, μl | Restriction condition |
   | --- | --- | --- |
   | FD 10x green buffer | 1.5 | 2 hours at 37 С |
   | KpnI | 0.5 |  |
   | XhoI | 0.5 |  |
   | TurboGFP C | 0.5 |  |
   | H2O | 12 |  |
   | **Total** | 15 |  |
4. Purify digested DNA from mixture.
5. Ligation:

   | Reagent | Volume, μl | Ligation condition |
   | --- | --- | --- |
   | 10x T4 liogation buffer | 1.5 | Overnight at 16 C |
   | T4 ligase | 0.5 |  |
   | LacZ | 10 |  |
   | TurboGFP C | 3 |  |
   | H2O | 0 |  |
   | **Total** | 15 |  |
6. Transformation (read more here)
7. Screening colonies:

   | Reagent | Volume, μl | PCR program |
   | --- | --- | --- |
   | 5x screen mix | 3 | 1. 95 C → 3' 29X: |
   | F-lacZ | 0.5 | 2. 95 C → 30'' |
   | R-lacZ | 0.5 | 3. 60 C → 30'' |
   | RNAse | 0.1 | 4. 72 C → 1:45'' |
   | H2O | 11 | 5. 72 C → 5' |
   | **Total** | 15 | 6. 12 C → inf |
   | **DNA** | 0 |  |
8. Growth positively screened colonies in LB overnight.
9. Use Miniprep to obtain plasmid DNA.
10. Confirm obtained DNA by using PCR:

    | Reagent | Volume, μl | PCR program |
    | --- | --- | --- |
    | 5x screen mix | 3 | 1. 95 C → 3' 29X: |
    | F-lacZ | 0.5 | 2. 95 C → 30'' |
    | R-lacZ | 0.5 | 3. 54 C → 30'' |
    | H2O | 10.5 | 4. 72 C → 1:45'' |
    | **DNA** | 0.5 | 5. 72 C → 5' |
    | **Total** | 15 | 6. 12 C → inf |

![](https://static.igem.org/mediawiki/2021/1/13/T--MIPT_MSU--Gag_Cloning.svg)

This protocol shows how we cloned gag into lentiviral vector.

**Materials:**

1. Gag plasmid from Laboratory of Cell Signalling Regulation.
2. Primers:

   F-gag - ATGCTCTAGAATGGGTGCGAGAGCGTC

   R-gag - ATGCGCGGCCGCTTATTGTGACGAGGGGTCGCTG
3. Evrogen Taq polymerase and its buffer.
4. Lentiviral vector plasmid.
5. Restriction: XbaI, NotI.
6. Thermo Fisher FastDigest x10 buffer.
7. T4 DNA ligase, 10x T4 ligation buffer.
8. Competent cells (XL10 Gold).

**Procedure:**

1. Restriction:

   | Reagent | Volume, μl | Restriction condition |
   | --- | --- | --- |
   | FD 10x green buffer | 1.5 | 2 hours at 37 С |
   | NotI | 0.5 |  |
   | XbaI | 0.5 |  |
   | Gag | 10 |  |
   | H2O | 2.5 |  |
   | **Total** | 15 |  |

   | Reagent | Volume, μl | Restriction condition |
   | --- | --- | --- |
   | FD 10x green buffer | 1.5 | 2 hours at 37 С |
   | NotI | 0.5 |  |
   | XbaI | 0.5 |  |
   | Vector | 0.5 |  |
   | H2O | 12 |  |
   | **Total** | 15 |  |
2. Purify digested DNA from mixture.
3. Ligation:

   | Reagent | Volume, μl | Ligation condition |
   | --- | --- | --- |
   | 10x T4 liogation buffer | 1.5 | Overnight at 16 C |
   | T4 ligase | 0.5 |  |
   | Gag | 10 |  |
   | Vector | 3 |  |
   | H2O | 0 |  |
   | **Total** | 15 |  |
4. Transformation (read more here)
5. Screening colonies:

   | Reagent | Volume, μl | PCR program |
   | --- | --- | --- |
   | 5x screen mix | 3 | 1. 95 C → 3' 29X: |
   | F-gag | 0.5 | 2. 95 C → 30'' |
   | R-gag | 0.5 | 3. 60 C → 30'' |
   | RNAse | 0.1 | 4. 72 C → 1:45'' |
   | H2O | 11 | 5. 72 C → 5' |
   | **Total** | 15 | 6. 12 C → inf |
   | **DNA** | 0 |  |
6. Growth positively screened colonies in LB overnight.
7. Use Miniprep to obtain plasmid DNA.
8. Confirm obtained DNA by using PCR:

   | Reagent | Volume, μl | PCR program |
   | --- | --- | --- |
   | 5x screen mix | 3 | 1. 95 C → 3' 29X: |
   | F-gag | 0.5 | 2. 95 C → 30'' |
   | R-gag | 0.5 | 3. 60 C → 30'' |
   | H2O | 10.5 | 4. 72 C → 1:45'' |
   | **DNA** | 0.5 | 5. 72 C → 5' |
   | **Total** | 15 | 6. 12 C → inf |

![](https://static.igem.org/mediawiki/2021/b/b3/T--MIPT_MSU--Gold_Transformation.svg)

Guidelines to transform E.coli XL-10 gold chemically competent cells.

**Materials:**

1. Chemically competent Cells (100 μl.)
2. Plasmid (around 100ng) or Ligation Mix.
3. LB.
4. Selective antibiotic:

   Amp

   Carb

   Kan

**Procedure:**

1. Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins).
2. Add plasmid/ligation mix, gently mix with cells, leave for 20-30 minutes in ice.
3. Heat-shock 30" at 42 С.
4. Put the tubes back on ice for 2 min.
5. In sterile conditions: add 500 ul LB to cells.
6. Keep shaking incubator at 37 C for 30' (Amp/Carb) or 1 hour (Kan).
7. In sterile conditions: Plate transformation onto 10 cm LB agar plate containing the appropriate antibiotic.
8. Incubate plates at 37 C overnight.

![](https://static.igem.org/mediawiki/2021/c/cd/T--MIPT_MSU--Gibson_Assembly.svg)

We used standard Gibson Assembly Cloning Kit, with protocol E5510
(more information from NEB can be found here) to construct 5 plasmids:

5’ - psi(short) - IRES - GFP - lacZ -3’

5’ - psi(short) - GFP -lacZ - 3’

5’ - psi(long) - IRES -GFP - lacZ - 3’

5’ - psi(long) - GFP - lacZ -3’

5’ - IRES - GFP - lacZ - 3’

Primers used:

|  |  |
| --- | --- |
| 1,2-vec-R | CGAGTCCTGCGTCGAGAGAGGACCGGTAGCGCTAGCG |
| 1,2-psi(s)-F | ATCCGCTAGCGCTACCGGTCCTCTCTCGACGCAGGACTCG |
| 1-psi(s)-R | CCACTGTTTAACGGTCTTCAGACGCTCTCGCACCCAT |
| 1-IRES-F | TGAAGACCGTTAAACAGTGGACGTTACTGGCCGAAGCC |
| 1,3,5-IRES-R | TCGTCGCTCTCCATGGTGGCTATTATCATCGTGTTTTTCAAAGGAAAACCACG |
|  |  |
| 2-psi(s)-R | CACTGTTTCCAACGGTCTTCAGACGCTCTCGCACCCAT |
| 2-vec-F | TGAAGACCGTTGGAAACAGTGGGTCGCCACCATGGAGAGC |
| 3,4-vec-R | CTGGTCTAACCAGAGAGACCGACCGGTAGCGCTAGC |
| 3,4-vec-R | CTGGTCTAACCAGAGAGACCGACCGGTAGCGCTAGC |
| 3,4-psi(l)-F | ATCCGCTAGCGCTACCGGTCGGTCTCTCTGGTTAGACCAGATCTGAG |
| 3-psi(l)-R | CCACTGTTTAACGGTCTTCATACTGACGCTCTCGCACCC |
| 3-IRES-F | TGAAGACCGTTAAACAGTGGACGTTACTGGCCGAAGCC |
| 4-psi(l)-R | CCAGGTGTTGCAAAGCTGGGATACTGACGCTCTCGCACCC |
| 4-vec-F | TCCCAGCTTTGCAACACCTGGGCCACCATGGAGAGCGAC |
| 5-vec-R | GCGGCTTCGGCCAGTAACGTGACCGGTAGCGCTAGCG |
| 5-IRES-F | ATCCGCTAGCGCTACCGGTCACGTTACTGGCCGAAGCC |

Numbers: 1, 2, 3, 4, 5 are the number of constructions in which primers are used.
Names: vec, psi(s), psi(l), IRES are target for Primers. Vec - vector, psi(s) - psi short, psi(l) - psi long.
F/R stands for Forward/Reverse.

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|| Fragment length: | Vector DNA length: |
| --- | --- |
| IRES - 551 nt | GFP + lacZ - 7754 nt |
| psi-long - 126 nt |  |
| psi-short - 356 nt |  |

The required mass of fragments for all constructions, except 3rd, was calculated using the NEBioCalculator.

For the 3rd construction, we had to double the molecular weight to increase the chances of success.

|  | Fragment mass, ng | Fragment mass, ng |
| --- | --- | --- |
| Psi-long | 13.77 | 0.4 |
| IRES | 21.32 | 0.5 |
| Linearized vector plasmid | 75 | 0.6 |
| Gibson Assembly Master Mix (2x) | - | 10 |
| Deionized Water | - | 8.7 |
| Total Volume (μl) |  | 20 |

Then we transformed our chemically competent cell (XL10 gold).

# Nucleofection and Vesicle treatment

We used Lonza 4D-NucleofectorTM X Unit(<https://bioscience.lonza.com/lonza_bs/GB/en/Transfection/p/000000000000203685/4D-Nucleofector-X-Unit>) with standard protocol(<https://bioscience.lonza.com/lonza_bs/RU/en/download/product/asset/21792>).

Nucleofection was performed on the MCF7 cell in a 6 well plate.

To the 5 of these wells 3 plasmids we added:

1. Syncytin(with stop codon)
2. gag-polyprotein
3. **one** of the constructions **obtained in a previous step**

   5’ - psi(short) - IRES - GFP - lacZ -3’

   5’ - psi(short) - GFP -lacZ - 3’

   5’ - psi(long) - IRES -GFP - lacZ - 3’

   5’ - psi(long) - GFP - lacZ -3’

   5’ - IRES - GFP - lacZ - 3’).
4. To the last well we added only control GFP plasmid from the kit.

Two days after nucleofection, when we observed the luminescence, we collected the supernatant containing vesicles from the wells and added it to the petri dishes with HT-29 cells (confluence 80%). Total of 5 petri dishes were used, each filled with a unique supernatant. After a single day of waiting we collected plates to observe the luminescence.

[![](https://static.igem.org/mediawiki/2021/c/c9/T--MIPT_MSU--arrow_top.svg)](#top)

[Syncytin cloning](#SynCytin_Cloning)

[LacZ cloning](#LacZ_cloning)

[Gag cloning](#Gag_cloning)

[E. coli XL-10 gold transformation](#Gold_Transformation)

[Gibson Assembly®](#Gibson_Assembly)

[Nucleofection and Vesicle treatment](#Nucleofection_and_Vesicle_treatment)

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

# Team:MIPT MSU/Results

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[Description](https://2021.igem.org/Team%3AMIPT_MSU/Description)[Engineering
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[human
practices](https://2021.igem.org/Team%3AMIPT_MSU/Human_Practices)
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[Human
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[Experiments and
protocols](https://2021.igem.org/Team%3AMIPT_MSU/Experiments)[results](https://2021.igem.org/Team%3AMIPT_MSU/Results)

Results

# Introduction

The development of our team is based on three main elements, which were assembled separately in three different plasmids.

The first element is the **endogenous retroviral protein syncytin** (ERVW-1 gene), which provides targeted fusion
of vesicles with cells that have a receptor for this protein (ASCT2).

The second element is the **target mRNA** with the **HIV-1 packaging signal** that needs to be delivered
to the cancer cell. The packing signal allows the sort mRNA to be packaged into a vesicle. As a target mRNA,
we used TurboGFP mRNA (Evrogen - [1]) with b-galactosidase (lacZ) gene, taken from a plasmid kit supplied
to us by the iGEM (Plate 2, well: 9E). Thanks to such a sensor, it is possible not only to fix the GFP
luminescence, but also to carry out blue-white selection. In other words, this fusion protein allows double control.

Last but not least element of our system is **gag-polyprotein**, which includes the RNA-binding domain (RBD).
This domain recognizes the psi-signal from the second type of constructs, dragging the sensory protein GFP + lacZ along with it.

These three elements, working together, carry out targeted packaging and delivery of vesicles to target cells.

# First type of genetic construction - fusion protein syncytin

Initially, we tried to obtain **syncytin from the MCF-7 cell culture**, in which its expression is observed.
We isolated total RNA from MCF-7 cells and performed reverse transcription followed by PCR for the ERVW-1 gene.
Using electrophoresis, we validated the result of obtaining cDNA of the ERVW-1 gene from MCF-7 cells and
three fragments of different lengths were found, one of which resembled the expected length of ERVW-1.
We assumed that this is due to the fact that our primers also annealed some other sequences present in
the MCF-7 culture, which were also present in the total cDNA. In this regard, it was decided to cut a
band corresponding to the length of ERVW-1 from the gel and repeat PCR. However, in the next round of
PCR, we also observed three bands corresponding to the same lengths. We believe that this is due to poor
separation of these three fragments on the gel, due to which some part of the non-target two genes nevertheless,
when excised from the gel, got into the excised piece of gel and was re-amplified. Due to this failure,
**we decided to order the synthesis of the syncytin gene** from Twist Bioscience. Having obtained this gene,
we repeated PCR and obtained the required fragment.

As part of the first plasmid, we decided to make two designs. One of the constructs contains a stop
codon at the end of the syncytin gene, while the other does not contain this stop codon, and after syncytin
comes the Turbo-GFP gene [1] in the syncytin reading frame. Thus, in the first case, we get pure syncytin,
and in the second case, a fusion protein consisting of syncytin at the N-terminus and Turbo-GFP at the C-terminus.
We cloned these two constructs and confirmed the success of the cloning by electrophoresis.

First type of genetic construction.

![](https://static.igem.org/mediawiki/2021/b/b7/T--MIPT_MSU--gen_map_1_1.png)

The gel electrophoresis results after Syncytin amplification from
cDNA obtained from the MCF-7 cell line. a)Top band (weight between 1500 and 2000 bp) potentially present correctly
amplified Syncytin (expected weight 1617 bp). b)Presents off target band with molecular weight around 1000 bp. c)
Presents off target band with molecular weight around 500 bp. Left column presents a 1kb+ Marker from Evrogen.

![](https://static.igem.org/mediawiki/2021/8/85/T--MIPT_MSU--1st_type_%283_bands%29_1.png)

The gel electrophoresis results after Syncytin amplification from
sequence synthesised by Twist Bioscience. Sample 1 - syncytin amplified by using primers with stop codon.
Sample 2 - syncytin amplified by using primers without stop codon. Left column presents a 1kb+ Marker from Evrogen.

![](https://static.igem.org/mediawiki/2021/b/b9/T--MIPT_MSU--1st_type_%28with%29_1.png)
![](https://static.igem.org/mediawiki/2021/c/cb/T--MIPT_MSU--1st_type_%28without%29_1.png)

# Second type of genetic construction - psi-signal with mRNA

The second construction contains a Ψ (psi) signal with the target mRNA (as already mentioned,
this is Turbo GFP and b-galactosidase mRNA). Initially, we planned to make one construct here, taking psi-signal
from lentiviral plasmids and merging it with the target mRNA. However, after careful consideration we concluded
that it is necessary to make 5 structures (read more here), shown in the figure below.

By using them we will be able to show that it is psi-signal that ensures the packaging of the target
mRNA into vesicles (however, looking ahead, we did not have time to do these experiments due to the compressed
amount of time). We ordered the synthesis of the psi-long gene from Evrogen. All structures of the second type
were assembled using Gibson Assembly. We confirmed the success of the assembly of the structures by electrophoresis.

Second type of genetic construction.

![](https://static.igem.org/mediawiki/2021/b/ba/T--MIPT_MSU--psi_2.png)

The gel electrophoresis results of 5 construction made with Gibson Assembly.
Left to right: 1kb+ Evrogen Marker, psi(short)-IRES-GFP-lacZ 1295 bp, psi(short)-GFP-lacZ 747 bp, psi(long)-IRES-GFP-lacZ 1525bp,
psi(long)-GFP-lacZ 973bp,IRES-lacZ 1149 bp. All constructions presented by bright white bands.

![](https://static.igem.org/mediawiki/2021/1/17/T--MIPT_MSU--2nd_type_1.png)

# Third type of genetic construction - gag-polyprotein

The last construcion is a lentiviral plasmid containing gag-polyprotein. This gag-polyprotein,
as mentioned at the beginning, includes RBD, to which the psi-signal packing signal is attached.
The lentiviral plasmid was chosen as the vector, since we want to carry out a stable transfection of MCF-7.
The gag-polyprotein gene was kindly provided by our instructor Evgenii Lunev (Institute of Gene Biology,
Russian Academy of Sciences). Successful cloning was also confirmed by agarose gel electrophoresis.

Third type of genetic construction.

![](https://static.igem.org/mediawiki/2021/5/52/T--MIPT_MSU--gag_construction_2.png)

The gel electrophoresis results after gag amplification after
cloning it into a lentiviral vector. Left column presents 1kb+ Marker from Evrogen. Right column presents
an amplified gag (weight 1503 bp).

![](https://static.igem.org/mediawiki/2021/6/61/T--MIPT_MSU--3rd_type_2.png)

Due to lack of time for the experimental part (due to the long supply of reagents to Russia,
difficulties with syncytin cloning and problems with plasmid extraction kit) we did not have time to do
the rest of the planned experiments, but we are not stopping here and will continue working on this project
after iGEM-2021. What else we plan to do is described below.

First, following the advice of Marlin company and our Instructor Ivan Sorokin, we want to carry
out in-vitro translation and transcription of the second type of our construction. This will allow us to check
the overall operation of our system outside the cells and confirm the concept of our work. It will also
confirm or deny that the presence of IRES in front of the target mRNA improves protein expression from it.

Second, we plan to transfect the target cells with the plasmids we have obtained. We plan to carry out transfection
into MCF-7 cells by electroporation using the Lonze kit. Two days after nucleofection we plan to collect a supernatant
containing vesicles and drip it onto HT-29. We expect that after such treatment, GFP and lacZ, which are signaling
proteins in our system, will be expressed in HT-29 cells.

Third, we will clean our vesicles with the help of columns and select such protocol parameters
so that the vesicle output will be more purified.

Finally, to improve our system, we plan to make a knockdown of the ASCT2 syncytin receptor in MCF-7 cells,
which are vesicle producers in our experiment. Thus, we exclude the possibility of reuptake by MCF-7 cells of
vesicles, which are budded from themselves.

We also plan to change the GFP+lacZ signaling mRNA to miRNA, with which we will be able to regulate the expression
of gene of interest. However, we will be able to start this part only after we confirm that our system works on signaling mRNA.
In addition, we plan to replace syncytin with other fusion proteins and thus change the targeting of our vesicles to other cell types.

# References

1. https://evrogen.com/products/TurboGFP/TurboGFP\_Detailed\_description.shtml
2. https://www.researchgate.net/figure/Caspase-activation-induced-by-different-drugs-on-HT-29-cell-line-was-evaluated-by-the\_fig5\_232249360

[![](https://static.igem.org/mediawiki/2021/c/c9/T--MIPT_MSU--arrow_top.svg)](#top)

[Introduction](#Introduction)

[First type of genetic construction - fusion protein syncytin](#First)

[Second type of genetic construction - psi-signal with mRNA](#Second)

[Third type of genetic construction - gag-polyprotein](#Third)

[References](#References)