iGEM 2024 — Team barcelona-ub

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Notebook

The laboratory notebook is a record of everything that is carried out in the laboratory. It is very important when it comes to monitoring the project, especially in our case, since we have carried out experiments in two different laboratories at the same time. The document below shows our team's laboratory notebook.

## HP Handbook

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Engineering

## Reneurish Engineering Overview

---

Reneurish aims to restore lost neuron activity in ischaemic stroke patients. To do so, we propose the use of genetically-modified neural progenitors enriched with neurotrophic factor expression. This expression is accomplished through transfecting iPSC-derived NPC with custom-designed vectors.

To fulfil our goal, we followed the Design-Build-Test-Learn (DBTL) cycle. It is a cornerstone in every synthetic biology project and Reneurish was no exception, these cycles have been fundamental to the development of our project, enabling us to design genetic circuits tailored to tackle specific needs.

We have divided our engineering strategy into three main Branches:

**Branch 1: Constitutive RFP Reporter Cassete**, where we tested the efficiency of two different construct designs to use mCherry as a transfection reporter. This led to *Branch 1.2: Customizable Transfection Reporter Cassette*, where we developed a chassis that future iGEM teams can use to customize reporters and their cellular localization.

Once we obtained the first results from Branch 1, we parallelly started with **Branch 2: Time-Dependent Neurotrophic Factor Overexpression**, for this we needed to determine the effects of overexpressing BDNF, the neurotrophic factor of choice, on neural activity. After verifying the positive effects of BDNF, we continued with *Branch 2.2: Time-dependant promoters to regulate BDNF expression*. In this stage, we are testing how different promoters regulate BDNF expression over time and how this impacts neural activity.

Finally, **Branch 3** is our way of looking towards the future. This branch includes design of future steps in Reneurish, which includes AAV infection of iPSC-derived NPC, the universalisation of our cell line via CRISPR-Cas Knock-Out of HLA phenotypes, and the addition of a co-transfection cassette to test cell proliferation in order to avoid the development of teratomas.

![](https://static.igem.wiki/teams/5316/images/atg-synbio-recovered-este.png)

![](https://static.igem.wiki/teams/5316/images/este-branca-3.png )

### Design and Building Strategy

We have used Type IIS cloning to assemble all constructs during the span of the project.

Type IIS restriction enzymes are a class of restriction enzymes (RE) that cut DNA a short distance away from their recognition site, rather than within the recognition sequence itself. Another interesting feature is that they generate sticky ends, which can be customizable and used to easily assemble multiple parts in a single reaction. [1]

Except in some justified cases to facilitate testing, the constructs have been transfected into Long-Term Self-Renewing Neuoepithelial Stem (lt-NES C4) derived from UBI001-A hiPSCs

These lt-NES cells are compromised to differentiate into neural progenitors, with restricted potential to become neurons, astrocytes, or oligodendrocytes. Differentiation is restricted under the exposure of WNT3A and BMP4 factors during the cortical priming process. Moreover, lt-NES cells are easily cryopreserved in liquid nitrogen with specific media for stem cells without DMSO.

This is useful for us because we can de-freeze and differentiate them into the cell type that best suits our needs. Also, lt-NES cells are capable of long-term self-renewal allowing for extended experiments. [2]

However, lt-NES cells require specific, delicate and more expensive working measures, which can difficultate culturing and maintenance. Therefore, clonings were done with XL-Blue bacteria and some of the tests were performed on HEK293T cells, which are feasibly transformed and have a rapid growth[3].

## Branches

---

Branch 1

Branch 1.2

Branch 2

Branch 2.2

Branch 3

## References

---

[1] Marillonnet S, Werner S. Assembly of Complex Pathways Using Type IIs Restriction Enzymes. Methods Mol Biol. 2019;1927:93-109. doi: 10.1007/978-1-4939-9142-6\_7. PMID: 30788787.

[2] Falk, A., Koch, P., Kesavan, J., Takashima, Y., Ladewig, J., Alexander, M., Wiskow, O., Tailor, J., Trotter, M., Pollard, S., Smith, A., & Brüstle, O. (2012). Capture of neuroepithelial-like stem cells from pluripotent stem cells provides a versatile system for in vitro production of human neurons. PloS one, 7(1), e29597. [https://doi.org/10.1371/journal.pone.0029597](%E2%80%9Dhttps%3A//doi.org/10.1371/journal.pone.0029597%E2%80%9D)

[3] Tan, E., Chin, C. S. H., Lim, Z. F. S., & Ng, S. K. (2021). HEK293 Cell Line as a Platform to Produce Recombinant Proteins and Viral Vectors. Frontiers in bioengineering and biotechnology, 9, 796991. [https://doi.org/10.3389/fbioe.2021.796991](%E2%80%9Dhttps%3A//doi.org/10.3389/fbioe.2021.796991%E2%80%9D)

[4] Zhang Y, Looger LL. Fast and sensitive GCaMP calcium indicators for neuronal imaging. J Physiol. 2024 Apr;602(8):1595-1604. doi: 10.1113/JP283832. Epub 2023 Mar 14. PMID: 36811153.

[5] Howe, E. S., Clemente, T. E., & Bass, H. W. (2012). Maize histone H2B-mCherry: a new fluorescent chromatin marker for somatic and meiotic chromosome research. DNA and cell biology, 31(6), 925–938. [https://doi.org/10.1089/dna.2011.1514](%E2%80%9Dhttps%3A//doi.org/10.1089/dna.2011.1514%E2%80%9D)

[6] Graham, F. L., Smiley, J., Russell, W. C., & Nairn, R. (1977). Characteristics of a human cell line transformed by DNA from human adenovirus type 5. Journal of General Virology, 36(1), 59-74. [https://www.researchgate.net](%E2%80%9Dhttps%3A//www.researchgate.net%E2%80%9D)

[7] Thomas, P., & Smart, T. G. (2005). HEK293 cell line: a vehicle for the expression of recombinant proteins. Journal of Pharmacological and Toxicological Methods, 51(3), 187-200. [https://www.sciencedirect.com](%E2%80%9Dhttps%3A//www.sciencedirect.com%E2%80%9D)

[8] Stepanenko, A. A., & Dmitrenko, V. V. (2015). HEK293 in cell biology and cancer research: phenotype, karyotype, tumorigenicity, and stress-induced genome-phenotype evolution. Gene, 569(2), 182-190. [https://www.sciencedirect.com](%E2%80%9Dhttps%3A//www.sciencedirect.com%E2%80%9D)

[9] Baldi, L., Hacker, D. L., Adam, M., & Wurm, F. M. (2007). Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives. Biotechnology Letters, 29(5), 677-684. [https://www.researchgate.net](%E2%80%9Dhttps%3A//www.researchgate.net%E2%80%9D)

[10] Shaw, G., Morse, S., Ararat, M., & Graham, F. L. (2002). Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells. The FASEB Journal, 16(8), 869-871. [https://www.researchgate.net](%E2%80%9Dhttps%3A//www.researchgate.net%E2%80%9D)

[11] Ventura, A., Meissner, A., Dillon, C. P., McManus, M., Sharp, P. A., Van Parijs, L., Jaenisch, R., & Jacks, T. (2004). Cre-lox-regulated conditional RNA interference from transgenes. Proceedings of the National Academy of Sciences, 101(28), 10380–10385. https://doi.org/10.1073/pnas.0403954101

[12] Flitsch, L. J., Börner, K., Stüllein, C., Ziegler, S., Sonntag-Buck, V., Wiedtke, E., Semkova, V., Au Yeung, S. W. C., Schlee, J., Hajo, M., Mathews, M., Ludwig, B. S., Kossatz, S., Kessler, H., Grimm, D., & Brüstle, O. (2022). Identification of adeno-associated virus variants for gene transfer into human neural cell types by parallel capsid screening. Scientific Reports, 12, 8356. https://doi.org/10.1038/s41598-022-12404-0

[13] Bernier, G. M. (1980). β2-Microglobulin: Structure, Function and Significance. Vox Sanguinis, 38(6), 323–327. [https://doi.org/10.1111/j.1423-0410.1980.tb04500.x](%E2%80%9Dhttps%3A//doi.org/10.1111/j.1423-0410.1980.tb04500.x%E2%80%9D)

[14] Amiri, F., Ranjbar, M., Pirouzfar, M., Nourigorji, M., & Dianatpour, M. (2021). HLA-A gene knockout using CRISPR/Cas9 system toward overcoming transplantation concerns. Egyptian Journal of Medical Human Genetics, 22(1), 37. [https://doi.org/10.1186/s43042-021-00155-y](%E2%80%9Dhttps%3A//doi.org/10.1186/s43042-021-00155-y%E2%80%9C)

[15] Chen, H., Li, Y., Lin, X., Cui, D., Cui, C., Li, H., & Xiao, L. (2015). Functional disruption of human leukocyte antigen II in human embryonic stem cell. Biological Research, 48, 59. [https://doi.org/10.1186/s40659-015-0051-6](%E2%80%9Dhttps%3A//doi.org/10.1186/s40659-015-0051-6%E2%80%9D)

[16] Gornalusse, G. G., Hirata, R. K., Funk, S. E., Riolobos, L., Lopes, V. S., Manske, G., Prunkard, D., Colunga, A. G., Hanafi, L.-A., Clegg, D. O., Turtle, C., & Russell, D. W. (2017). HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells. Nature Biotechnology, 35(8), 765–772. [https://doi.org/10.1038/nbt.3860](%E2%80%9Dhttps%3A//doi.org/10.1038/nbt.3860%E2%80%9D)

[17] Han, X., Wang, M., Duan, S., Franco, P. J., Kenty, J. H.-R., Hedrick, P., Xia, Y., Allen, A., Ferreira, L. M. R., Strominger, J. L., Melton, D. A., Meissner, T. B., & Cowan, C. A. (2019). Generation of hypoimmunogenic human pluripotent stem cells. Proceedings of the National Academy of Sciences, 116(21), 10441–10446. [https://doi.org/10.1073/pnas.1902566116](%E2%80%9Dhttps%3A//doi.org/10.1073/pnas.1902566116%E2%80%9D)

[18] Petrus-Reurer, S., Romano, M., Howlett, S., Jones, J. L., Lombardi, G., & Saeb-Parsy, K. (2021). Immunological considerations and challenges for regenerative cellular therapies. Communications Biology, 4(1), 1–16. [https://doi.org/10.1038/s42003-021-02237-4](%E2%80%9Dhttps%3A//doi.org/10.1038/s42003-021-02237-4%E2%80%9D)

[19] Hoerster, K., Uhrberg, M., Wiek, C., Horn, P. A., Hanenberg, H., & Heinrichs, S. (2021). HLA Class I Knockout Converts Allogeneic Primary NK Cells Into Suitable Effectors for “Off-the-Shelf” Immunotherapy. Frontiers in Immunology, 11. [https://doi.org/10.3389/fimmu.2020.586168](%E2%80%9Dhttps%3A//doi.org/10.3389/fimmu.2020.586168%E2%80%9D)

[20] Kagoya, Y., Guo, T., Yeung, B., Saso, K., Anczurowski, M., Wang, C.-H., Murata, K., Sugata, K., Saijo, H., Matsunaga, Y., Ohashi, Y., Butler, M. O., & Hirano, N. (2020). Genetic Ablation of HLA Class I, Class II, and the T-cell Receptor Enables Allogeneic T Cells to Be Used for Adoptive T-cell Therapy. Cancer Immunology Research, 8(7), 926–936. <https://doi.org/10.1158/2326-6066.CIR-18-0508>

[21] Mattapally, S., Pawlik, K. M., Fast, V. G., Zumaquero, E., Lund, F. E., Randall, T. D., Townes, T. M., & Zhang, J. (2018). Human Leukocyte Antigen Class I and II Knockout Human Induced Pluripotent Stem Cell-Derived Cells: Universal Donor for Cell Therapy. Journal of the American Heart Association, 7(23), e010239. [https://doi.org/10.1161/JAHA.118.010239](%E2%80%9Dhttps%3A//doi.org/10.1161/JAHA.118.010239%E2%80%9D)

[22] Xu, H., Wang, B., Ono, M., Kagita, A., Fujii, K., Sasakawa, N., Ueda, T., Gee, P., Nishikawa, M., Nomura, M., Kitaoka, F., Takahashi, T., Okita, K., Yoshida, Y., Kaneko, S., & Hotta, A. (2019). Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates iPSCs with Enhanced Immune Compatibility. Cell Stem Cell, 24(4), 566-578.e7. [https://doi.org/10.1016/j.stem.2019.02.005](%E2%80%9Dhttps%3A//doi.org/10.1016/j.stem.2019.02.005%E2%80%9D)

[23] Seminatore, C., Polentes, J., Ellman, D., Kozubenko, N., Itier, V., Tine, S., Tritschler, L., Brenot, M., Guidou, E., Blondeau, J., Lhuillier, M., Bugi, A., Aubry, L., Jendelova, P., Sykova, E., Perrier, A. L., Finsen, B., & Onteniente, B. (2010). The Postischemic Environment Differentially Impacts Teratoma or Tumor Formation After Transplantation of Human Embryonic Stem Cell-Derived Neural Progenitors. Stroke, 41(1), 153–159. [https://doi.org/10.1161/STROKEAHA.109.563015](%E2%80%9Dhttps%3A//doi.org/10.1161/STROKEAHA.109.563015%E2%80%9D)

[24] Kochurani, K. J., Suganya, A. A., Nair, M. G., Louis, J. M., Majumder, A., Kumar K., S., Abraham, P., Dutta, D., & Maliekal, T. T. (2015). Live detection and purification of cells based on the expression of a histone chaperone, HIRA, using a binding peptide. *Scientific Reports*, 5, 17218. [https://doi.org/10.1038/srep17218](%E2%80%9Dhttps%3A//doi.org/10.1038/srep17218%E2%80%9D)

[25] Basu, S., Campbell, H. M., Dittel, B. N., & Ray, A. (2010). Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS). Journal of Visualized Experiments: JoVE, 41, 1546. [https://doi.org/10.3791/1546](%E2%80%9Dhttps%3A//doi.org/10.3791/1546%E2%80%9D)

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[![](https://static.igem.wiki/teams/5316/images/cycle1.png)](#cycle1)
[![](https://static.igem.wiki/teams/5316/images/cycle2.png)](#cycle2)

# BRANCH 1: Constitutive RFP Reporter Cassette

---

Overall, the main goal is to determine the design that best works as a transfection reporter and that will be used throughout the rest of the constructs of the projects.

It consisted of two different cycles.

### Cycle 1: BDNF followed by mCherry

#### Design

The cell line used during the Reneurish project (UBI001-A hiPSCs cell line) has been genetically modified to express GCamP -a calcium detection system- on the GFP spectrum [4].

For this reason, when adding the transfection reporter for our constructs, we needed a reporter with a different wavelength. We chose mCherry because it is one of the most common Fluorescent Proteins, meaning there is a wide range of techniques and optimised protocols already at our disposal to test it. Additionally, mCherry's red emission spectrum is useful for multicolor imaging, as it can be used combined with other fluorescent proteins (like GFP) without significant spectral overlap.

In order to have mCherry as a transfection reporter, we wanted it to be regulated by the same promoter as BDNF in a polycistronic construct, so we used the following scheme:

![](https://static.igem.wiki/teams/5316/images/eng-canvi-design-c1.png)

#### Build

The construct was transfected using a Lentivirus vector into lt-NES cells before starting differentiation. We expected to start seeing expression on day 1 after transfection.

We used Lentiviral transfection to ensure we would be able to see the expression of our construct for an extended period of time. In this case, we infected lt-NES and wanted to see the expression signal up to the point where they were differentiated into neurons.

During this process, cell proliferation is notably elevated, thus we aimed to avoid episomal transfection, as this would result in exponential dilution of the expression signal. Therefore, the ability of the lentiviral vector to integrate the construct into the host genome was highly beneficial for maintaining stable expression and obtaining results.

#### Test

The goal of the experiments from this cycle was to know if the design fit the requirements to use RFP as a transfection reporter. The activity of the reporter was studied through fluorescence microscopy, measuring the signal from mCherry in the range of 550-650 nm wavelength. Additionally, we checked the GFP emission spectrum to make sure there were no interferences between fluorescent proteins.

For more information see **Page: Results**.

#### Learn

From this cycle we concluded:

* mCherry can be used as a reporter for our cell line because it does not overlap with the GFP emission spectrum and will not interfere with the Calcium detection system.
* The design of the construct worked, but it wasn’t useful as a transfection reporter as we intended it to be. We realized that since mCherry is a cytoplasmic protein, it complicated differentiating the limits between cells and identifying the transfected ones.

  ![](https://static.igem.wiki/teams/5316/images/learn-1-cycle-1.png)

  Why do we need to count individual cells?
  Why do we need to count individual cells?

  On further steps of our project (Branch 3), we want to transfect a genetic circuit into lt-NES cells with AVV and be able to select only the infected cells. Thus, we need to be able to differentiate which cells express the reporter.

  To solve this, we entered the next cycle with a new design.

### Cycle 2: Nucleus-targeted RFP.

To solve the problems that arose during cycle 1, we thought of building a nucleus-targeted reporter.

#### Design

To make sure that our reporter is located in the nucleus, easing the cell counting process, we designed a Composite Part (BBa\_K5316020) of H2B linked to the mCherry sequence

Why H2B?
Why H2B?

Histone H2B is a core component of the nucleosome, it is part of the histone octamer, around which DNA is wrapped.

In research, H2B is commonly fused to fluorescent proteins to guide these proteins to the nucleus [5]. This fusion takes advantage of H2B’s natural localization to chromatin, ensuring that the attached fluorescent proteins accumulate specifically in the nucleus.

This approach is particularly useful for visualising nuclear structures, tracking gene expression, or studying nuclear dynamics in live cells.

#### Build

We also changed the building strategy. The purpose of this cycle was to check if the new design containing H2B succeeded at locating mCherry into the nucleus with the goal of being able to differentiate those cells which had been transfected and expressed the construct.

To do this, we realised we could simplify the testing process by not using neurons derived from lt-NES, which required Lentiviral transfection, it is not an immortal cell line and incur higher costs due to their media and growth factor requirements, instead -to be more efficient- we thought of doing the tests on HEK293T cells.

Why HEK293T cells?
Why HEK293T cells?

The HEK293T cell line is derived from human embryonic kidney cells. These cells stand out for their favourable characteristics for laboratory procedures. Their robust nature allows them to grow easily in laboratory conditions [6]. Furthermore,they are characterised by their rapid growth, driven by its high proliferation rate [7]. This feature increases the efficiency of transfection, since dividing cells are more susceptible to the entry of DNA to the nucleus.

The high transfection rate is their best asset. In addition to all mentioned above, its high efficiency in this procedure can be attributed to its great capacity to internalise exogenous DNA, since the composition of its membrane favours the endocytosis of lipid complexes with DNA, which is what happens when transfection is performed with lipofectamine, for example. However, its versatility allows success with different types of transfections [8].

Moreover, these cells present a good intracellular environment for transcription and translation, which implies high protein expression of the transfected plasmid [9]. Finally, the relative undifferentiation of HEK293T makes them more prone to internalise exogenous DNA than other more differentiated cell lines, and also it seems there is a relationship between HEK293T cell line and the neuronal lineage [10].

Overall, doing the experiments on HEK293T cells will already give us the needed information to know if the construct works, and it will easen the transfection and data gathering process.

HEK293T cells will be transformed using lipofectamine 3000.

#### Test

We followed the same testing strategy as in Cycle 1, this way we will be able to compare results between cycles. The activity of the reporter was studied through fluorescence microscopy, measuring the signal from mCherry at in the range of 550-650 nm wavelength, and especially focusing on the cellular localization of the signal.

The goal of the experiments from this cycle was to know if this new design fit the requirements to use H2B-RFP as a transfection reporter.

#### Learn

Testing for this cycle is being carried out at this moment, results are expected to be ready during the judging session.

Based on literature reviews [5] we expect this genetic circuit to allow us to locate the transfected cells.

If results meet our expectations, we will be closing the loop for this first Branch of the project.

1. We needed a reporter that did not interfere with the calcium detection system.

2. We found mCherry successfully fit this requirement. However, the design did not serve as a transfection reporter system because it located the reporter throughout the cytoplasm rather than concentrated it in the nucleus, difficulting cell counting.

3. We redesigned the construct to target our reporter into the nucleus using H2B as a signal peptide, solving the counting cells problem.

![](https://static.igem.wiki/teams/5316/images/3loopstatic.png)

Branch 1 consisted of the cycles aimed at testing POC0. To learn more about POC0, **see: POCs Page**

← Overview

Next branch →

[![](https://static.igem.wiki/teams/5316/images/cycle3.png)](#cycle3)

[![](https://static.igem.wiki/teams/5316/images/cycle3-1.png)](#cycle3-1)
[![](https://static.igem.wiki/teams/5316/images/cycle3-2.png)](#cycle3-2)
[![](https://static.igem.wiki/teams/5316/images/cycle3-3.png)](#cycle3-3)

# BRANCH 1.2: Customizable Transfection Reporter Cassette

#### A Part Collection for future iGEM teams

As we worked with our construct, we realised that the use of reporters targeted to specific cellular locations could be highly helpful to other iGEM teams.

This led us to initiate Branch 1.2 with the goal to create a reporter cassette that allows its users to choose both the signal peptide and the linked reporter according to their preferences and the specific requirements of their projects.

### Cycle 3: Customizable Reporter Cassette

#### Design

Using our previous design from Cycle 2 as the foundation for Cycle 3, we implemented specific modifications to develop a chassis that allows the user to select and switch both the cellular region for reporter localization and the type of fluorescent protein they want to use

To do so, we thought of adding restriction enzyme’s recognition sites that flanked both the H2B and mCherry sequence.

![Cycle 3 design](https://static.igem.wiki/teams/5316/images/final-design-cycle-3.png)

The raw structure of this plasmid consists of two primary components: a signal peptide and a fluorescent protein. Each sequence is flanked by a strategically placed Type IIS restriction enzyme.

The final design on this cycle includes CMV as a constitutive promoter, followed by the H2B sequence, linked to mCherry, which are flanked by Esp3I and BsaI recognition site respectively.

#### Build

We needed to build both the backbone and the inserts. Since we planned to use Type IIS assembly as the cloning method, we needed to design the overhangs for each of our parts to properly fit the backbone, taking into consideration both the position and direction of each insert.

#### Backbone

#### Backbone

Our plasmid backbone functions as the chassis for the Customizable Transfection Reporter Cassette, designed to fit different genetic parts at distinct, specific positions. This required careful planning to ensure compatibility with different components.

Our goal was to design a cassette that allows users to modify either the signal peptide, the reporter gene, or both. To achieve this, we designed unique overhangs for the insertion sites of the signal peptide and the reporter gene, ensuring that each element could be assembled at its intended position without interference.

![Single Peptide assembly](https://static.igem.wiki/teams/5316/images/eng-sp-assembly.png)![Reporter assembly](https://static.igem.wiki/teams/5316/images/eng-reporter-assembly.png)

#### Inserts

#### Inserts

We generated a library of different Parts for commonly used Signal Peptides and Fluorescent Proteins, including the overhangs specifically adapted to fit the designated positions in the backbone, assuring compatibility.

Since our backbone has been designed to be compatible with the iGEM kit, we have been able to use Parts from previous iGEM teams and included them in the library. Additionally, to contribute more to the community, we have added three new Signal Peptides and two Fluorescent Proteins. This will allow teams to target new cellular locations with their reporters and to use new colours.

### Fluorescent Proteins

| **Kit number** | **FP** | **Part name** | **TIIS RE** | **Flanking compatibility** | **Uses** |
| --- | --- | --- | --- | --- | --- |
| 9 | eCFP | BBa\_E0020 | BsaI | AATG/GCTT | 95 |
| 10 | eYFP | BBa\_E0030 | BsaI | AATG/GCTT | 140 |
| 16 | TannenRFP | BBa\_J97003 | BsaI | AATG/GCTT | 1 |
| N/A | EGFP | BBa\_K1071009 | BsaI | AATG/GCTT | 1 |
| N/A | bfVFP | BBa\_K5316012 | BsaI | AATG/GCTT | - |
| N/A | SVFP | BBa\_K5316013 | BsaI | AATG/GCTT | - |

### Signal Peptides

| **Kit number** | **Localisation** | **Part name** | **TIIS RE** | **Flanking compatibility** |
| --- | --- | --- | --- | --- |
| N/A | Membrane | BBa\_K5316015 | Esp3I | ACGA/TCTC |
| N/A | Mitochondria | BBa\_K5316014 | Esp3I | ACGA/TCTC |
| N/A | Nucleous | BBa\_K5316007 | Esp3I | ACGA/TCTC |
| N/A | ER | BBa\_K5316017 | Esp3I | ACGA/TCTC |

It’s important to emphasise that all overhangs in this design can be easily added to other reporters, making them compatible with the cassette. This means that other teams can incorporate any fluorescent protein or signal peptide of their choice, even if they aren’t already included in this library.

For more info **see: Part Collection and FP Handbook**

#### Test

Assemblies for this cycle were validated using a cloning model (Geneious Prime). The goal was to check that the overhangs were correct and they allowed the insertion of the different fragments at the desired positions.

![Assembly test cycle 3](https://static.igem.wiki/teams/5316/images/assembly-test-cycle-3.png)

#### Learn

From this cycle we concluded:

* We confirmed that the assembly of H2B and mCherry into the desired positions of the backbone succeeded, meaning that we had correctly designed the Type IIS recognition site, cutting direction and overhangs.
* From these assemblies we saw that the Ampicillin Part we used was not compatible with the Type IIS cloning system because it contained a BsaI recognition site in the position 772.

During the learning step of this cycle, we acquired valuable insights and thought of multiple enhancements for our design. Consequently, we engaged in multiple iterations of the same cycle to refine our approach effectively:

#### Cycle 3.1

#### Cycle 3.1

#### Design

Derived from our learnings of the previous cycle, we needed to eliminate the BsaI recognition site to make it compatible with our cloning system.

The modification was done at the nucleotide level, so the final peptide remains the same as the unmodified ampicillin without affecting functionality.

#### Build

We changed GGTCTC nucleotides at position 723 for GAGATC.

![](https://static.igem.wiki/teams/5316/images/ampicilin-bsai.png)

#### Test

This modification has been proved to work because transformed E. coli have been successfully selected and grown in Amp+ media.

#### Learn

* We learned that the modification successfully adapts the Ampicillin sequence to be Type IIS compatible

Further experiments can be conducted to confirm the success of this new part. For example, we could digest the ampicillin sequence with BsaI and run the resulting fragments on an agarose gel. We would expect to see a single band, as BsaI should not find a recognition site to cleave, leaving the fragment intact.

* We also thought of adding an extra recognition site for a different Type IIS enzyme: BbsI flanking the whole cassette. This way different teams can insert the reporter cassette into their plasmids.

![](https://static.igem.wiki/teams/5316/images/cycle-3-1-learn.png)

* However, while revising the design and results for this cycle we thought of a way to improve it and make it easier for future iGEM teams to use it as a transfection reporter; instead of having mCherry at the reporter position we will add an ORF Stuffer, a new Part that has not been registered by any team before.

#### Cycle 3.2

#### Cycle 3.2

#### Design

Changing the mCherry sequence from the backbone for an ORF stuffer. This way, other teams can easily interchange it for whatever reporter better suits the needs of their project.

What is an ORF Stuffer
What is an ORF Stuffer

The ORF Stuffer, composed of amino acids 2-83 of E. coli β-galactosidase, functions as a negative open reading frame (ORF) control in molecular biology experiments. It is an innocuous peptide without any functionality.

This stuffer sequence is specifically designed to occupy vector space without generating a functional protein, enabling researchers to evaluate the effects of true gene expression without interference from background activity
See Part registry at: **BBa\_K5316010**

![](https://static.igem.wiki/teams/5316/images/cicle-3-2-disseny.png)

#### Build and Test

Using the same modelling system as in previous iterations, we confirmed the correct assembly of the ORF Stuffer into our backbone and verified that it generates the correct overhangs when digested with BsaI.

![](https://static.igem.wiki/teams/5316/images/build-and-test-cycle-3-2.png)

#### Learn

* Assemblies successfully work on the modelling program.

#### Assemblies Modelling Results

#### Assemblies Modelling Results

![](https://static.igem.wiki/teams/5316/images/cycle-3-2-bfvfp.png)
![](https://static.igem.wiki/teams/5316/images/cycle-3-2-egfp.png)

![](https://static.igem.wiki/teams/5316/images/cycle-3-2-eyfp.png)
![](https://static.igem.wiki/teams/5316/images/cycle-3-2-rfp.png)

![](https://static.igem.wiki/teams/5316/images/cycle-3-2-svfp.png)
![](https://static.igem.wiki/teams/5316/images/cylce-3-2-ecfp.png)

* Since all assemblies worked, we decided that by default, the raw backbone will include a nuclear localization signal (H2B) and an ORF stuffer in the position designated for the fluorescent protein (FP). For the sake of our project, it was essential that the reporter was directed to the nucleus, however, the user has the flexibility to change the localization signal in the same reaction where the FP is inserted, allowing easy customization.
* We realised we hadn’t checked if the changes of the different Parts, and the scars left by the digestion and ligation process, had altered the reading frame. We tackled this problem in the following iteration.

#### Cycle 3.3

#### Cycle 3.3

#### Design

We had the following starting parameters:

* After the Signal Peptide (SP), we had Esp3I’s recognition and restriction sites, which were 11 bp in length. Changing the SP would reduce Esp3I’s sites to a 4 bp scar.
* Following the linker, we had BsaI’s recognition and restriction sites, which were also 11 bp in length. Changing the ORF\_Stuffer to a Fluorescent Protein (FP) would reduce BsaI’s sites to a 4 bp scar.
![“Results”](https://static.igem.wiki/teams/5316/images/eng0.png)* ORF aberrations:
  + The untouched backbone had 22 additional bp, changing the ORF.
  ![“Results”](https://static.igem.wiki/teams/5316/images/enga.png)+ Only changing the ORF\_Stuffer to a FP would result in 15 additional bp, technically maintaining the ORF. However, those 15 bp are not distributed in codons in the two scarred regions, changing the ORF for the linker but maintaining it for the FPs.
  ![“Results”](https://static.igem.wiki/teams/5316/images/engb.png)+ Changing both the SP and ORF\_Stuffer would result in 8 additional bp, changing the ORF.

![“Results”](https://static.igem.wiki/teams/5316/images/engc.png)

Therefore, we needed to find a way to maintain the ORF of the linker and the FPs by only changing the ORF\_Stuffer or both SP and ORF\_Stuffer.

#### Build

After three design iterations, we came up with the perfect solution. We had to add a total of 6 bp in selected positions for the ORF to be maintained in all possible combinations:

* 1. **2 bp after the linker sequence but right before the BsaI site**. Since the ORF\_Stuffer is always changed for a FP, the BsaI restriction site is always reduced from 11 bp to a 4 bp scar. Therefore, adding 2 bp to the 4 bp scar allows for the maintenance of the ORF since there will now be 6 bp between the linker and the FP.
* 2. **2 bp after the Esp3I site and before the linker**. There are two options:
  ![“Results”](https://static.igem.wiki/teams/5316/images/engbuild2.png)

  + 2.a If the SP is changed, the scar will be 4 bp long. Therefore, adding 2 bp to the 4 bp scar allows for the maintenance of the ORF since there will now be 6 bp between the SP and the linker.
  ![“Results”](https://static.igem.wiki/teams/5316/images/engbuild2a.png)+ 2.b If the SP remains unchanged, there will be the 11 bp of the unmodified Esp3I site, and the 2 additional bp we have introduced. These 13 bp change the ORF.
  ![“Results”](https://static.igem.wiki/teams/5316/images/engbuild2b.png)
* 3. **2 bp after the H2B sequence and before the Esp3I site**. This corrects the ORF aberration in the case that the SP remains untouched and the ORF\_Stuffer is changed with a FP. Therefore, the number of nucleotides between the H2B and the linker is 15 (2 + 11 + 2), maintaining the ORF.

![“Results”](https://static.igem.wiki/teams/5316/images/engbuild3.png)

Lastly, we had to carefully choose which nucleotides we wanted to insert to minimise the effect of the additional aminoacids. In the end, we added the following residues:

* FP substitution: H2B - **[GVSSL]** - Linker - **[G]** - FP
* SP and FP substitution: SP - **[SL]** - Linker - **[G]** - FP

#### Test

Using the same modelling system as in previous iterations, Geneius, we confirmed that with the new adaptations of the design, none of the assemblies changed the reading frame.

#### Learn

* We were able to efficiently correct the changes of the fused ORFs by inserting just 6 bp in specific locations.
* We minimised the effect of the scars by choosing the aminoacids with the lowest steric hindrance
* In silico testing was successful.
* The next step is to translate the design to the laboratory

![](https://static.igem.wiki/teams/5316/images/2bp-santi.png)

Finally, we obtained the perfect design that fit all the requirements needed to create the Customizable Transfection Reporter Cassette, with a library of interchangeable Signal Peptides and Fluorescent Proteins adapted for other iGEM teams to use.

← Overview

Next branch →

[![](https://static.igem.wiki/teams/5316/images/cycle4.png)](#cycle4)

# BRANCH 2: Time-Dependent Neurotrophic Factor Overexpression

---

One of the main goals of the Reneurish project is to genetically modify neural progenitors to overexpress the neurotrophic factor BDNF and reduce the damage after an ischemic stroke event by improving neural activity regeneration.

**Learn more** about why we chose BDNF as the neurotrophic factor to express.

### Cycle 4: BDNF followed by mCherry

#### Design: Overexpression of Neurotrophic factors to increase neural activity

The main goal of this cycle is to check the effects of constitutive overexpression of BDNF on neural progenitors and the consequences on the numbers of active neurons, neural connectivity, cell maturation and population interactions.

To achieve this, we put BDNF under the regulation of EF1A, a constitutive eukaryotic promoter, followed by the transfection reporter chassis from Branch 1. Moreover, we flanked this reporter cassette with LoxP sites, enabling us to remove it once approaching the clinical steps of the project.

![](https://static.igem.wiki/teams/5316/images/periquito-electrocutat-4.png   )

#### Build

With the assistance of our PI, lt-NES cells were transfected with a Lentivirus.

Learn more about lentiviral transfection safety on **Page: Safe Lab Practices**.

Transfected cells were then differentiated via exposure to WNT3A and BMP4 factors at post infection.

#### Test

To check transfection and functionality of our construct and BDNF expression, we conducted both Immuno and histochemical assays and BDNF qPCR, respectively.

To assess different aspects affected by BDNF overexpression on lt-NES, we performed a variety of experiments:

* 1. Effects on differentiation: Immuno-histochemical assays and culture observation, focusing on differentiation markers such as PSD95, MAP2, NeuN, etc.
* 2. Effects on projection formation: Chip-cultures
* 3. Effects of synaptic activity: GECI calcium recordings.

#### Learn

Throughout the diverse experiments performed and comparing the different parameters between the control and transfected (tBDNF) cells, we could determine that overexpressing BDNF caused:

* Higher cell count in comparison to control cells, without generating uncontrolled growth.
* BDNF expressing cells generate more projections.
* BDNF expressing cell cultures contain more active neurons than the control.
* BDNF expression does not affect the formation of neuronal networks, avoiding aberrant networks.

See the results obtained from this tests on **Page: results**.

← Overview

Next branch →

[![](https://static.igem.wiki/teams/5316/images/cycle5.png)](#cycle5)

# BRANCH 2: Time-Dependent Neurotrophic Factor Overexpression

---

One of the main goals of the Reneurish project is to genetically modify neural progenitors to overexpress the neurotrophic factor BDNF and reduce the damage after an ischemic stroke event by improving neural activity regeneration.

Learn more about why we chose BDNF as the neurotrophic factor to express.

### Cycle 5: Time-dependant promoters to regulate BDNF expression

#### Design

Once we confirmed the beneficial effects of BDNF on neural activity with Cycle 4, we entered Cycle 5. In this cycle, we innovated by replacing the constitutive EF1A promoter in the design and adding pA, pB, and pC\*. These new promoters are time-dependant, which will allow us to determine which one is the best to express BDNF only during the first stages of the therapy. Our goal is to compare their regulatory capacity to determine which achieves the most effective BDNF levels.

\*Promoter names are censored under a patent pending system. To learn more, see **Page: IP**.

Additionally, these promoters are inactivated once neuronal differentiation is complete, providing an extra layer of clinical safety. See more info in **Page: Clinical Safety**

![](https://static.igem.wiki/teams/5316/images/cycle-5-design.png)

#### Build

Promoters are assembled in the same construct as in Cycle 4, where they replace EF1a. By using different flanking restriction enzymes, the promoters are integrated with the proper orientation in the vector.

#### Test

A double digestion was carried out to ensure that both the plasmid backbone and the promoters had the required cohesive ends. The digestion was verified through an electrophoresis gel and afterwards the ligation reaction was prepared with a insert : vector ratio of 5 : 1. To validate the ligation, a second digestion was performed using enzymes that cut once in the backbone and once within the ligated promoters, but not in the control.

This method allowed us to easily determine if the correct promoters had been inserted into the backbone:

* Two bands: This indicates that the enzyme cut twice—once in the backbone and once in the promoter of interest.
* One band: This indicates that the enzyme only found a single recognition site in the backbone, meaning the construct contains the control promoter.

All tested plasmids were linearized, indicating they contained the original EF1a promoter

#### Learned

Through the weeks spent in the laboratory, we learned the importance of meticulous preparation, validation, and optimising experimental conditions to ensure efficient and reliable lab outcomes.

With the results from Cycle 5 we learnt that:

* The promoters of interest were not ligated in the vector as we have proven in the gel electrophoresis (see **Page: Results, Plasmid assembly**).
* Unwanted DNA fragments may be the limiting factor in our assembly.
* DNA gel recovery after a complete digestion may allow us to succeed assembling the plasmid.

Learn more about how we are trying to overcome this on **Page: Results, Plasmid assembly**

![](https://static.igem.wiki/teams/5316/images/fig-1-gel-electrophoresis-to-check-ligation-1-jpg.png)

![](https://static.igem.wiki/teams/5316/images/fig-2-gel-electrophoresis-to-check-ligation-2.png)

← Overview

Next branch →

[![](https://static.igem.wiki/teams/5316/images/cycle-branch-3-incomplete.png)](#cycle6)

# BRANCH 3: Designs for integrated future steps

## From the lab bench to the patient

---

Branch 3 is our way of looking towards the future. This branch includes design of future steps in Reneurish, which includes AAV infection of iPSC-derived NPC, the universalisation of our cell line via CRISPR-Cas Knock-Out of HLA phenotypes, and the addition of a co-transfection cassette to test cell proliferation in order to avoid the development of teratomas.

### Branch 3.1: AAV transfection

#### Design: Overexpression of Neurotrophic factors to increase neural activity

Using engineered cells for cell therapies requires strict safety measurements. This is why we are interested on using AAV to transfect our NPC, as they do not integrate into the cell genome and their expression disappears overtime.
For more information regarding use of adeno-associated viruses for cellular therapy,
**See Page: Biosafety.**

#### Build

From the very beginning, we have optimised our BDNF expression construct for use in AAV. By using an AAV-compatible backbone and introducing loxP sites to optimise sequence length for encapsulation.

#### LoxP removal

Our BDNF expression vector contains a reporter cassette with an mCherry-H2B fusion protein. However, our final product cannot contain fluorescence-emitting cells, which is why from day one our construct has LoxP sites flanking the DNA sequence of the reporter cassette.
By designing our LoxP sites orientated in the same direction inside the plasmid, we intend to generate a deletion in our expression plasmid that removes our reporter cassette [11].

#### AAV serotype

Selecting the serotype of the AAV proteic capsid can help improve transfection efficiency, as well as increase cell specificity.
We intend to use AAV7m8 for our transfections, as it is the best performing serotype in iPSC transfection in comparison to other AAV serotypes [12].

#### Cell transfection

Transfection of our cells with AAV will be performed during the differentiation (priming) stage. This ensures transfection is performed at optimal time of cell proliferation, and prevents overdilution of the adenovirus before patient transplant.

### Branch 3.2: Cell universalisation

In the future, the Reneurish strain will undergo a HLA Knock-Out modification to universalise our cell line.

One of the main holdbacks of cell therapies are immunogenic rejection towards the grafted cells. For Reneurish to reach as many patients as possible, we need to find a way to overcome this issue.

In a nutshell, an immunogenic rejection towards the grafted cells occurs when host cytotoxic T cells recognise foreign HLA antigens, activating Natural Killer Cells (NKs). These, in turn, recognise the HLA marker that does not belong to the host, triggering a response to terminate the cells containing this mismatched antigen [13].

After researching different options, we have decided to universalise our cell line via CRISPR/Cas9 by eliminating the HLA genes from our cell line. This essentially creates non-immunogenic cells.

HLA-KO is of special interest in cell therapies and has been performed on numerous occasions [14, 15, 16, 17, 18] due to the possibility of evading T-cell responses.

Not only that, but a combination of Knocked-Out genes and a Knock in allows the cells to bypass recognition by Natural Killer cells [19, 20].

#### Design

Using engineered cells for cell therapies requires strict safety measurements. This is why we are interested on using AAV to transfect our NPC, as they do not integrate into the cell genome and their expression disappears overtime.
For more information regarding use of adeno-associated viruses for cellular therapy,
**See Page: Biosafety.**

#### Knock-out

According to literature, with a simple 3 gene Knock-Out we might be able to bypass allogenic detection. The genes we are interested in Knocking-Out are:

* **β2-Microglobulin (B2M):**These genes codify proteins that allow HLA class I proteins to be presented on cell surface [21]. Eliminating the expression of this protein prevents the presentation of HLA-I molecules on cellular surface. This KO allows us to bypass cytotoxic T-cell detection.
* **MHC class II TransActivator (CIITA):**Is the master regulator for HLA-II gene expression [22]. Knock out of this gene blocks transcription of HLA-II genes, giving a second layer to avoid an allogenic response.
* **T-cell Receptor Alpha Constant (TRAC):**These genes codify the T-cell receptors (TCRs). We would specifically target the TCR-⍺ region to completely abolish TCR expression [14].

To Knock-Out these genes, we will design single guide RNAs (sgRNAs) for the CRISPR/Cas9 system to introduce Double Strand Breaks in the target genes. To properly knock out the genes, we will prepare two sgRNAs per gene, cutting at different points and introducing large deletions to completely Knock-Out expression.

#### Knock-in

In order to avoid triggering the Natural Killer response, we need to knock in the expression of polymorphic HLA-E molecules. The recognition of HLA-E by the NK cells avoids induced “missing-self” lysis of the transplanted cells.

As shown in literature, the ideal knock in is a fusion protein consisting of [20]:

* HLA-E single-chain dimer (heavy chain)
* B2M

The proteins are covalently fused via a flexible linker.

Knock in will be performed via lentiviral transfection to ensure the modification of the cell strain persists through future generations.

### Branch 3.1: AAV transfection

Safety is key for Reneurish. This is why we want to implement a specialised cell selection method using Flow-cytometry.

After transfection of our NPC cells with AAV to express BDNF, the cells have to undergo strict testing to minimise any risk of teratoma formation. This means we need a highly efficient method capable of selecting cells individually.

This new construct will be co-transfected with our BDNF expression circuit, meaning it will also behave as our transfection reporter once we have removed the fluorescence cassette from the backbone via LoxP sites.

#### Design

#### Expression

According to a study conducted in embryonic stem cell-derived Neural Progenitors, the likelihood of teratoma formation oscillates according to the maturation stage of the transplanted cells [23]. To minimise teratoma formation, we need to transplant NPC in their Mid to Late stage of maturation, which is why we have chosen SOX2, a Neural Progenitor Cell specific promoter for this cassette.

#### Marker

Controlled by the SOX2 promoter will be the expression of a custom synthetic membrane protein not expressed in any other cell types [24]. This protein will be recognised by a specific fluorescent antibody so we can perform Fluorescence-Activated Cell sorting, which has purity rates of 95 to 100% [25].

By adding fluorescence to the cell surface, we are not hindering cell viability nor manipulating them, and the superficial expression of the fluorophore makes the cells compatible for the FACS system.

For clinical application, the transplanted cells need to be highly purified and strictly tested. The FACS sorting process allows us to select cells individually and not by population, diminishing risk of teratoma formation and allowing selection of BDNF-expressing cells.

For additional safety, we might implement back-to-back rounds of FACS sorting of the cells to minimise any potential error.

← Overview

{% endblock %}


{% extends "layout.html" %}
{% block page\_content %}

* [Transfection reporter](#one)
* [Effect of BDNF on NPC](#three)
* [BDNF expression](#six)
* [Plasmid Assembly](#tortuga)
* [References](#ten)

Results

### Overview

After 3 months of lab work, our experiment data has allowed us to reach the following conclusions:

* Cytoplasmic RFP is a successful transfection reporter in iPSC-derived NPC, however it does not allow cell counting.
  Our nuclei-bound mCherry reporter successfully allows selection of transformed cells.
* tBDNF and mBDNF cells present higher speeds in differentiation rates in comparison to control cells.
* tBDNF cells present higher cell count compared to control cells without generating uncontrolled growth.
* BDNF expressing cells generate more projections than mBDNF and WT cells.
* BDNF expressing cell cultures contain more active neurons than the control.
* BDNF expression does not affect the formation of neuronal networks.
* BDNF expression levels are 7 times the basal expression of actin.
* Promoter ligation was not achieved, however we have identified the origin of the error and will work on it in the following weeks.

## Transfection reporter

---

Our lentivirus had a cytoplasmic transfection reporter (RFP). Using staining 1 we amplified RFP’s signal by using an RFP-antibody bound to Cy3. We successfully observed RFP fluorescence in transfected cells and none in WT cells.

![ ](https://static.igem.wiki/teams/5316/images/results-1.png)
![ ](https://static.igem.wiki/teams/5316/images/results-2.png )

As we can observe, the only red found in the WT samples are noise particles, validating that our cells were successfully transfected and that our reporter works. Moreover, there is no cross-talk between the red fluorescent channel and the green fluorescent channel, which makes RFP (mCherry) a viable fluorescent reporter for our GCamP-GFP expressing cell line.

## Nuclei counting

When comparing the use of the RFP-cytoplasmic reporter with the nuclei reporter DAPI, we confirmed our assumptions that this transfection reporter would not allow us to accurately count the transfected cells.

![ ](https://static.igem.wiki/teams/5316/images/results-3.png)

The cytoplasmic expression of RFP is distorted across all the cells in the culture and doesn’t have defined edges that allow us to count the cells for proliferation assays or obtain transfection efficiency rates.

Seeing these results, it is clear that we need a different transfection reporter method for our cell line and future experiments.

## Transfection and proof

We have transfected HEK293T cells with our H2B-mCherry construct, and have successfully confirmed nucleus-reported fluorescence.

![](https://static.igem.wiki/teams/5316/images/mcherru-nuc-1.webp)
![](https://static.igem.wiki/teams/5316/images/mcherru-nuc-2.webp)
![](https://static.igem.wiki/teams/5316/images/mcherru-nuc-3.webp)

The localised fluorescence in the cell nuclei allows us to easily identify the transfected cells, meaning that out construct is a great tool to select only transformed cells.

![](https://static.igem.wiki/teams/5316/images/mcherry-nuc-merged1.webp)
![](https://static.igem.wiki/teams/5316/images/mcherry-nuc-merged2.webp)
![](https://static.igem.wiki/teams/5316/images/mcherry-nuc-merged3.webp)

By overlaying the red fluorescence layer with the 2D images of the cell culture, we are able to visualise how fluorescence falls within each cell, demonstrating that the reporter is in the nuclei.
We can also see a difference between untransfected cells and transfected cells, as the fluorescence isn’t found in all cells. This was to be expected, as lipofectamine transfection doesn’t have a very high efficiency. This makes our reporter a great choice to select transformed cells from WT.

## Effect of BDNF on NPC

---

Assessing the effect of BDNF on our NPC cultures is essential to Reneurish.

We have performed a variety of experiments to test the effect of BDNF-expression on different neural characteristics:

* **Differentiation:** Immunohistochemical assays and culture observation.
* **Projection formation:** Chip-cultures.
* **Synaptic activity:** GECI calcium recordings.

## Differentiation

BDNF has been shown to promote differentiation and maturation of neural progenitor cells [1]. This is why we wanted to see how it affected our NPC cultures.

## The experiment

To test and determine the differentiation and maturation levels in our cell cultures, we took progress pictures via simple microscopy, as well as performed immunohistochemical assays.

These assays would use antibodies to mark different proteins and transporters characteristics of neurons and astrocytes, that would also help determine the cells’ maturity.

In essence, we have 4 different types of markers according to what information they provide of the cultures:

* Transfection reporter: RFP
* Synaptic markers: PSD95, SynI
* Neuronal markers: MAP2, NeuN, TujI
* Astrocyte marker: GFAP

For more information on the staining composition:

| Staining | Primary | Secondary | Color |
| --- | --- | --- | --- |
| Staining 1 | anti-RFP (Mouse) | Anti-Mouse | CY3 (red) |
|  | anti-PSD95 (Rabbit) | Anti-Rabbit | CY5 (magenta) |
|  | anti-MAP2 (Chicken) | Anti-Chicken | 488 (green) |
|  | DNA | DAPI (blue) |  |
| Staining 3 | anti-SynI (Mouse) | Anti-Mouse | CY3 (red) |
|  | Anti-NeuN (Rabbit) | Anti-Rabbit | CY5 (magenta) |
|  | Anti-GFP (Goat) | Anti-Goat | 488 (green) |
|  | DNA | DAPI (blue) |  |
| Staining 4 | Anti-PSD95 (Mouse) | Anti-Mouse | CY5 (magenta) |
|  | Anti-MAP2 (Chicken) | Anti-Chicken | 488 (green) |
|  | Anti-GFAP |  | CY3 (red) |
|  | DNA | DAPI (blue) |  |

For further information regarding the functions of the proteins, consult **this** section.

## Culture Evolution

Routine microscopy observations of our cultures allowed us to observe physiological changes of our neurons and oscillations in cell confluency.

### Pre-transfection

The first images were taken on May 29th, right before lentiviral transfection and 13 days after thawing the It-NES cells.

![ ](https://static.igem.wiki/teams/5316/images/results-4.png )
![ ](https://static.igem.wiki/teams/5316/images/results-5.png )

At this stage, cells were still in a very early stage of development but already started presenting neural-like characteristics, such as the association of cell bodies in rosettes.
We can also observe the cells starting to generate projections, but it is still too early to tell whether they will become axons or dendrites.

![ ](https://static.igem.wiki/teams/5316/images/project-clump1.jpg)

### Post-transfection

The day after transfection (May 30th), no significant changes in cell confluence or survival were observed.

![ ](https://static.igem.wiki/teams/5316/images/results-6.png)
![ ](https://static.igem.wiki/teams/5316/images/results-7.png)

This means that the cell transfection has not affected cell viability.

### Day 16

On day 16 post-differentiation, we observed the progress of our cultures. The confluence of the cells is much greater, and the clusters formed by the neural bodies have become much larger.

We can also observe how the size of the neural projections, such as the axons, have extended significantly, some even seeming to connect different neural rosettes.

![ ](https://static.igem.wiki/teams/5316/images/results-8.png)

## Immunohistochemical Assays

### Cell Quantification

Cell quantification allows us to see whether BDNF promotes neural survival or affects cell count. This also helps us observe whether constitutive expression of BDNF generates uncontrolled growth and presents risk of teratomas.

To quantify the number of cells per image, we isolated the DAPI (blue) layer marking the cell nuclei, and used imageJ for automatic cell counting.

This process required all images to be inverted and converted to 8-bit. We then increased contrast by adjusting the threshold to reduce system error. We automatised the process by using the Image-based Tool for Counting Nuclei (ICTN) plugin. We used the same settings for all counting, using 6 px as the set diameter and a minimum distance of 8 px.

#### Day 16: Control and tBDNF

By day 16, the amount of cells is greater in tBDNF cultures than control cultures, and the size and depth of the neural aggregates is also much greater in tBDNF cultures. This is a positive sign, as bigger neural aggregates are a sign of higher differentiation.

On day 16 post-differentiation we fixed a control ibidi plate and a tBDNF ibidi plate.

The biggest noticeable difference between these two cultures is the size of the neuronal aggregates and the width of the different cultures. The confocal microscope we used to take the images analyses the cultures and takes pictures at different Z levels (different depth planes), and whilst the control culture needed 3 planes, the tBDNF culture needed 7.

![ ](https://static.igem.wiki/teams/5316/images/results-9.png)
![ ](https://static.igem.wiki/teams/5316/images/results-10.png)

We estimate that the cell count of these images might present a significant error due to the images containing large neuronal clusters. This most likely affects the tBDNF culture most, as the depth of the culture is much greater.

Nonetheless, the program estimated that the control plate contained 6398 cells, whilst the tBDNF plate contains a total of 9236 cells . In this case, the tBDNF culture has 30% more cells than its WT counterpart. However, due to the size of the neural aggregates, the estimation is not entirely reliable.

For further count imaging, we will try to select areas of the plates with the least possible amount of neural aggregates to obtain more reliable data.

#### Day 52: Control, mBDNF, and tBDNF

Day 52 plates contain much more mature cells and stable cultures. In this case, cells from both stain 3 and stain 4 ibidi cells were counted using the automated method mentioned previously. We observed how, on average, tBDNF cultures contain 80% more cells than their control counterparts.

![ ](https://static.igem.wiki/teams/5316/images/results-11.png)
![ ](https://static.igem.wiki/teams/5316/images/results-12.png)
![ ](https://static.igem.wiki/teams/5316/images/results-13.png)
![ ](https://static.igem.wiki/teams/5316/images/results-14.png)
![ ](https://static.igem.wiki/teams/5316/images/results-15.png)

Control cell count is within the 1010-960 interval, averaging 989 cell nuclei per ibidi plate. In comparison, tBDNF count belongs in the 1640-1860 interval, and averages 1781 . This means that tBDNF cells have around 800 more cells per plate than the control cells.

However, the difference in cell count is not great enough to suggest highly proliferative cells nor uncontrolled growth, meaning that despite an increase in cell number, the risk of teratoma formation is very low.

![ ](https://static.igem.wiki/teams/5316/images/results-graphic-2.jpg)

### Differentiation

Stain 1 was used to visualise maturity during the early stages of differentiation (day 16), whilst stains 3 and 4 allowed us to tell maturity at later stages (day 52)

#### Early stages: stain 1

At early stages (day 16 post-differentiation), we observe neuron differentiation markers, but we don’t find PSD95, a mature synaptic marker.

![ ](https://static.igem.wiki/teams/5316/images/results-16.png)
![ ](https://static.igem.wiki/teams/5316/images/results-17.png)

There is no expression of PSD95 (magenta) in neither control nor tBDNF cultures, meaning the cells are still in an early stage of development. No significant differences are observed between control and tBDNF cells.

#### Late stages: stain 3

![ ](https://static.igem.wiki/teams/5316/images/results-18.png)
![ ](https://static.igem.wiki/teams/5316/images/results-19.png)

The presence of synapsin is a key marker of mature synaptic formations. By contrasting the images, we clearly observe how the red fluorescence intensity of synapsin is higher in BDNF expressing cells than control cells. This might be an indicator that BDNF positively affects the formation of synapses between neurons. We will confirm with our GCamP recording analysis, which you can find below .

We quantified NeuN by isolating the NeuN fluorescent layer, automatised the counting process using Image J. We inverted the images, transformed them to 8 bit, and adjusted the threshold to 100. We then used the Image-based Tool for Counting Nuclei (ICTN) plugin at 8px diameter and 30px minimum distance

![ ](https://static.igem.wiki/teams/5316/images/results-20.png)
![ ](https://static.igem.wiki/teams/5316/images/results-21.png)

We calculated the proportion of NeuN expressed in the cells by normalising the cell count by the number of cell nuclei detected in the cell, which we’d obtained previously from our cell quantification.

The results show that the tBDNF culture doubles the control culture in proportion of NeuN expressing cells. Whilst 3,86% of control cells express NeuN, 7,86% of tBDNF cells are NeuN expressing. This means that NeuN is found two-fold in tBDNF cultures in comparison to the control.

NeuN, being often used as a late differentiation marker, confirms that BDNF-expressing cells are proportionally more mature in comparison to the negative control cells.

![ ](https://static.igem.wiki/teams/5316/images/results-graphic-3.jpg)

#### Late stages: stain 4

Higher presence of PSD95 in both tBDNF and mBDNF cells in comparison to control cells allows us to confirm that BDNF favours the speed of neuronal differentiation.
This staining also allowed us to verify that our NPC are able to differentiate into astrocytes in addition to neurons.

![ ](https://static.igem.wiki/teams/5316/images/results-22.png)
![ ](https://static.igem.wiki/teams/5316/images/results-23.png)
![ ](https://static.igem.wiki/teams/5316/images/results-24.png)

Differences in PSD95 (Pink) allow us to confirm that BDNF favours cell differentiation. As we can observe, PSD95 staining in BDNF cells (mBDNF, tBDNF) highlights the axons, demonstrating presence of the post-synaptic channel. In contrast, control cells’ axons are not stained with Cy5, and we mostly observe the stain in the cell body.

The increase of PSD95 in cells under exposure to BDNF helps us cement that it speeds up neural differentiation and maturation.

This is the only stain we have marked astrocytes with GFAP. This was interesting as our NPC are supposed to differentiate into all neural cell types, not only neurons. In these images, and clearly standing out in red, we were able to verify that our iPSC-derived NPC are able to produce more than one neural cell type.

## Projection Formation

The generation of projections by the different cell types is analysed via the chip culture plates assay. By quantifying cells on each side of the channels and counting the amount of axons entering and exiting them, we determined that BDNF exposure helps improve the formation and extension of projections, in comparison to WT cells.

## Stain

These chips were all stained with the staining 2 solution. We decided to implement the staining two solution to better visualise the neurons, as it contains markers for both mature (MAP2) and immature (Tuj1) neuron markers stained in green (488). This gives a more universal view of the neurons present in the culture, and ensures visualisation of all neurons.

| Staining 2 | | | | |
| --- | --- | --- | --- | --- |
| anti-NeuN | Rabbit | Anti-Rabbit | Cy5 (magenta) |
|  | anti-MAP2 | Mouse | Anti-Mouse | 488 (green) |
|  | anti-tubulin | Mouse |  |  |
|  | DNA |  |  | DAPI (blue) |

## Axon count

Axon counting was performed on images with the entire plane of the chip plates, with a total of 120 channels per plate.

Whilst tBDNF and mBDNF cells emit at least 1 axon per channel, control cells only emit 2 axons per every 5 channels . This confirms that BDNF exposure to cells, whether in media or by cell expression, increases the amount of axons generated by neurons.

![ ](https://static.igem.wiki/teams/5316/images/results-graphic-4.jpg)

Due to the presence of plastic from the plate separating the channels, counting of the axons that cross in the middle of the channels is not recommended. Primarily as interference from the fluorescence and light dispersion is not picked up by the microscope, but also because due to the small size of the channels the antibody solution struggles to flow through and stain the axons. This is why we counted the axons that were entering the channels from either sides of the cell compartments instead of the axons crossing. This is also a more reliable method, as in the channels axons get compacted and can’t be counted adequately.

![Image 1](https://static.igem.wiki/teams/5316/images/results-25.png)
![Image 2](https://static.igem.wiki/teams/5316/images/results-26.png)
![Image 3](https://static.igem.wiki/teams/5316/images/results-27.png)

It is important to note, that despite the control chip having nuclei close to the axon channels, this has not translated to the presence of MAP2 and Tuj (green stain). As observed in the image, very few green projections are found close to the channel entrances, which may have affected the low results of the control chip.

In contrast, mBDNF had a very high density of axons close to the channels, generating the opposite problem. The high density of axons and their association might have resulted in overestimating the amount of axons that enter the channels. This specially affected the entering axons on the left side of the chip.

|  | Left | Right | Total |
| --- | --- | --- | --- |
| WT | 28 | 48 | 76 |
| tBDNF | 55 | 140 | 195 |
| mBDNF | 148 | 96 | 244 |

The “right” value for each respective chip plate corresponds to WT cells for the control chip, tBDNF cells for the tBDNF chip, and mBDNF cells for the mBDNF chip. In contrast, the “left” value always corresponds to WT cells.

We were pleasantly surprised by the increase in emitted projections by the BDNF-expressing neurons in comparison to the positive control (mBDNF). Not only that, but the tBDNF cells have generated three times the projections than their WT counterpart.

Taking into account how the mBDNF cells have also emitted more projections than the WT cells, we can confirm that BDNF exposure positively affects the emission of projections by neurons, and that constitutive expression by said neurons is preferred over media exposure to BDNF.

This means that our transfected neurons are better suited for integration into brain tissue than neurons treated with BDNF.

## Synaptic Activity

To study our cells’ synaptic activity, we performed multiple recordings of GFP expression bound to GCamP, which allowed us to visualise changes in action potential, and therefore when and where synapses were taking place.

We used the program HoKaWo from Hamamatusu to perform the recordings, and analysed the data with Netcal and custom MatLab scripts.

![ ](https://static.igem.wiki/teams/5316/images/recording1.gif)
![ ](https://static.igem.wiki/teams/5316/images/recording2.gif)

## ROI Selection and individual Calcium spikes

In order to collect and interpret the data from the culture recordings, we used Netcal program to identify active cells (ROI), by detecting changes in brightness intensity.

This program generates traces of individual fluorescent intensity of each ROI, and visualises them into a spike-like map. Each spike represents an action potential of the cell, detected by an increase and decrease in fluorescence.

![ ](https://static.igem.wiki/teams/5316/images/spikes.png)

The spike profiles of the different neuronal cultures present normal patterns, meaning that no frequency aberrations are observed in presence of BDNF.

### Active cells

Thanks to analysing the amount of active cells via their synaptic activity, we were able to determine that expression of BDNF increases the amount of active neurons in culture.

An additional feature of Netcal is quantifying the amount of active cells per video. We have grouped together the data from early-stage recordings and late-stage recordings and compared results to determine whether the effect of BDNF expression affects the number of active neurons.

![ ](https://static.igem.wiki/teams/5316/images/geci-recording.png)

As shown in the diagram, the number of active cells in tBDNF (purple) cultures doubles the amount of active control (orange) neurons. This pattern is not very significant at early stages of differentiation, but increases with neuron maturation. .

## Community synapses

The analysis of community synapses takes into account the global interaction of neurons with each other in the establishment of networks.
We have observed how BDNF exposure does not negatively affect the formation of the neuronal network nor does it generate aberrations, meaning that the functionality of the networks stays the same as the control.

Due to size differences between tBDNF and control communities, the following data only takes into account synaptic activity, but not cell amount.
For these analysis, we had to downsize the tBDNF sample so that the number of active tBDNF cells matched the number of active control cells in order to avoid introducing any bias to the data. In our particular case, we had to downsize the sample size to 75 cells for both control and tBDNF cultures.

***Analysis - Network connectivity maps***

What we have observed is that the differences in synaptic networks are negligible. tBDNF cells are establishing the same networks as their control counterparts. The size of the communities is variable, but it is not a significant parametre.

The network connectivity maps use the information derived from the adjacency connectivity matrices by connecting all the cells that have established a connection in a 3D space.
This allows us to “see” the shape of the synaptic network to assess whether there are any significant changes in their composition. The different colours of the graph represent the neurons belonging to different communities.

![ ](https://static.igem.wiki/teams/5316/images/raster-plot.png)

These results allow us to confirm how expression of BDNF in neurons does not change the composition of synaptic networks, meaning that BDNF does not have a negative effect in their constitution.

***Raster Plot***

From the spike maps obtained from each individual ROI, the program extracts a raster plot, which represents every single active cell within the recording, were the blue dots represent when they experience an active potential, namely, when they synapse.

Each horizontal line represents a cell, and the blue dots that make it up represent the timepoint during the recording at which they synapse. Blue dots belonging to the same vertical line are synapses that occur at the same time, and are a sign of cell communication, specially if the behaviour is presented repeatedly.

***Adjacency connectivity matrices***

Adjacency connectivity matrices are derived from the raster plots. The raster plot matrix vertical axis, which is used to identify individual neurons, is positioned in both the vertical and horizontal axis.
In this case, the black dots in the matrix represent an effective connection established between two different neurons (or, in other words, a simultaneous synapse).
This analysis allows us to define neuron communities, which are shaded in different colours.
This has allowed us to observe that whilst control cultures have larger and fewer cultures, tBDNF cultures have generated more neuron communities but of smaller size.

***Other network parameters***

These parameters were calculated using MATLAB’s Brain Connectivity Toolbox [2]

**GEFF\_TH:**
Global efficiency accounts for the capacity of neurons to exchange information across the entire network.

When Gₑ ≃ 0, the ROI poorly communicate with each other, whilst Gₑ ≃ 1 indicates higher capacity of information exchange throughout the global network.

![ ](https://static.igem.wiki/teams/5316/images/geff-th.png)

**Leff\_TH:**
Local efficiency is a similar measurement to Global efficiency, where the neuronal connection is judged by singular nodes instead of the community.

![ ](https://static.igem.wiki/teams/5316/images/leff-th.png)

**Q\_TH:**
Modularity index account the tendency of neurons to form functional modules.

In essence, it evaluates wether the neurons of a community have higher tendency to generate connections within their own community, or whether they have a greater tendency to reach out to other communities.

High Q values indicate low connectivity between different neuron communities, whilst low Q values indicate high connectivity within the same community.

![ ](https://static.igem.wiki/teams/5316/images/q-th.png)

**Comm\_Size\_5more:**
Community size calculates the relative size of the different neuronal communities.

![ ](https://static.igem.wiki/teams/5316/images/comm-size-5more.png)

**SmallWorldness\_SW:**
Small Worldness is a parameter used to compare internal organisation of the neuronal communities.

![ ](https://static.igem.wiki/teams/5316/images/smallworldness-sw.png)

**Ave\_L:**
Ave\_L helps quantify the number of connexions established between neurons.

![ ](https://static.igem.wiki/teams/5316/images/ave-l.png)

Overall, averages of the aforementioned parameters show no significant differences in magnitude. This means that BDNF-expressing cells in culture do not alter the generation and interactions of the neuronal networks nor the neuronal communities.

## Conclusions

---

The different assays we have performed have allowed us to determine that BDNF expression in iPSC-derived NPC has great potential for neural transplants.

Not only do BDNF expressing NPC differentiate at a slightly faster rate than WT NPC, but they also generate cultures with higher neuron counts, without generating excessive growth (teratomas).

We have also confirmed that BDNF expressing cells are able to emit a high number of projections, indicating that these cells can easily integrate into brain tissue, facilitating the incorporation of the transplanted cells into the patients’ brains.

Not only that, but BDNF has a positive effect in neurons by increasing the neuronal activity without affecting the composition of the neuronal network nor generating aberrations.

## Constitutive BDNF expression

---

In order to determine the magnitude of BDNF expression under constitutive promoter EF1a, we extracted cell culture media from control and BDF-expressing cells at multiple points during the experiment to perform a qPCR assay.

**qPCR data**

| qPCR analysis Reneurish | | | |
| --- | --- | --- | --- |
 Conditions | Average Experimental Ct Value (BDNF) | Average Experimental Ct Value (GAPDH) | Average Experimental Ct Value (Actin) || Conditions | Testing Gene (BDNF) | Housekeeping Gene (GAPDH) | Housekeeping Gene (Actin) |
| WT | 26,03444917 | 23,90796934 | 21,52323232 |
| BDNF | 30,46665792 | 31,29373045 | 31,58479845 |
| deltaCt Value | | | |
| (BDNF-GAPDH) | (WT-GAPDH) | (BDNF-Actin) | Value (WT-Actin) |
| -0,827072525 | 2,126523829 | -1,118140528 | 4,51116995 |
|  | ΔΔCt GAPDH | ΔΔCt Actin |  |
|  | -2,953596354 | -5,629310478 |  |
|  | 2^(-ΔΔCt) | 2^(-ΔΔCt) |  |
|  | 7,746777769 | 49,49841686 |  |

**qPCR analysis**

Using the actin housekeeping gene as reference, we can determine that BDNF is expressed sevenfold in comparison to the control (WT) cells.
This means that the constitutive expression of BDNF under EF1a promoter is 7 times the basal expression of actin, whilst the wild-type presents no BDNF expression.

![ ](https://static.igem.wiki/teams/5316/images/qpcr-graph.png )

## Plasmid Assembly

---

We want to test BDNF expression under three different promoters. We ordered one construct that contains EF1a constitutive promoter, so our plan was to digest the construct to remove EF1a, and then ligate the different promoters into the backbone.

We are currently working on the ligation step as we have obtained inconclusive results.

## Ligation verification

To verify that the ligation had occurred as expected, we used Sanger sequencing to sequence the promoter region of the plasmid, where the different gene fragments (promoters) should have inserted. Unfortunately, the results obtained from the sequencing service were ambiguous and hindered the possibility to draw definitive conclusions.

Instead, to verify ligation we designed a new digestion protocol of the plasmid using new enzymes followed by a gel electrophoresis. In essence, in presence of the desired promoter (successful ligation), we would obtain two bands in the gel. If the desired promoter wasn’t in the plasmid, we would obtain 1 single band of the uncut plasmid.

In all cases the first restriction site is found in the plasmid backbone and the second one is found inside the promoters pA, pB and pC but not in EF1a. Thus, if the promoter of interest has been ligated to the vector two bands should appear in the gel. Otherwise, only one band will appear. In the following table we can see the sizes of the expected bands for each situation:

| Digestion | Sizes of Expected Fragments (if Successful Ligation) | Sizes of Expected Fragments (if Original Plasmid) |
| --- | --- | --- |
| Digestion to check for pA ligation | 6150 bp + 850 bp | 8000 bp |
| Digestion to check the pB ligation | 700 bp + 6800 bp | 1000 bp + 7000 bp |
| Digestion to check the pC ligation | 6250 bp + 950 bp | 8000 bp |

## Digestion results

After the digestion, we ran 1% agarose gels and loaded 2 replicas of each promoter ligation. Plasmids are named pVB432-(+inserted promoter + replica nº + “ ‘ ” if their ligation is still not validated). For each of the plasmid replicas, we loaded a digested sample (“+ enz”) and a not digested replica as control.

![](https://static.igem.wiki/teams/5316/images/fig-1-gel-electrophoresis-to-check-ligation-1-jpg.png)
![](https://static.igem.wiki/teams/5316/images/fig-2-gel-electrophoresis-to-check-ligation-2.png)

For the expected inserted promoters A, B and C we always see the same pattern:

* Digested sample: a single band between 7 kb and 10 kb.
* Control sample: a single band bigger than 10 kb.

This pattern is not the expected 2 bands always smaller than 7 Kb for promoters A, B and C so the expected ligation has not occurred.

Moreover, the digested band size is around 8 Kb, which matches the size of the opened pVB432-EF1a vector. The DNA in the control band appears to be bigger than 8 kb, a plasmid size that is bigger than any of pVB432-EF1a, pVB432-A, pVB432-B or pVB432-C vectors. The explanation for this result is that circular plasmids migrate differently than linearised plasmids and depending on several factors, this migration is slower than the linearised form of the plasmid. [3]

Taking into consideration that we decided not to purify only the DNA fragments of interest, it makes sense that the results indicate that our ligated vectors contain mainly EF1a and not our promoter sequences.

Lastly, before designing a new approach for our plasmid assembly we tried to contemplate other possible limiting factors for the ligation. Initially only a 1% agarose gel had been run to check the ligation (see figure 3). This step was enough to validate the pVB432-EF1a was effectively digested but there was not enough resolution to conclude the same for the promoter's digestion (labelled as A, B and C in the Figures 3 and 4). Therefore, we ran a 4% agarose electrophoresis gel with the digested and control samples of the promoters. There is little resolution for promoter B, as it is the biggest of all (close to 800 bp) and the two digested fragments are about 20bp on each side of the promoter, so a 40bp difference is barely noticeable. Regarding A and B promoters, the digested vs control size difference can be clearly noticed.

![](https://static.igem.wiki/teams/5316/images/fig-3-gel-electrophoresis-to-check-digestion-1.png)
![](https://static.igem.wiki/teams/5316/images/fig-4-gel-electrophoresis-to-check-digestion-2.png)

We conclude that the limiting factor of our assembly lies in the preparation of the ligation reaction: while the digested DNA fragments of interest are successfully obtained during the double digestion, the ligation reaction contains an excess of unwanted fragments.

Aiming to overcome the latter issue our next steps in the lab are the following:

* Repeat the double digestion step ensuring the conditions to obtain a complete digestion of the DNA.
* After digestion, proceed with a gel electrophoresis DNA recovery of the promoters and vector bands to ensure that we are **only** adding the desired DNA in the ligation reaction.
* Set several ligation reactions at different insert:vector ratios to find the most optimal.

## References

---

[1] Gibson, E. G., Oviatt, A. A., & Osheroff, N. (2020). Two-Dimensional Gel Electrophoresis to Resolve DNA Topoisomers. Methods In Molecular Biology, 15-24. https://doi.org/10.1007/978-1-0716-0323-9\_2

[2] Rubinov, M., & Sporns, O. (2010). Complex network measures of brain connectivity: Uses and interpretations. NeuroImage, 52(3), 1059–1069. https://doi.org/10.1016/j.neuroimage.2009.10.003

[3] Waterhouse, E. G., An, J. J., Orefice, L. L., Baydyuk, M., Liao, G.-Y., Zheng, K., Lu, B., & Xu, B. (2012). BDNF Promotes Differentiation and Maturation of Adult-born Neurons through GABAergic Transmission. Journal of Neuroscience, 32(41), 14318–14330. https://doi.org/10.1523/JNEUROSCI.0709-12.2012

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