iGEM 2022 — Team duke

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

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

## Inspirations

---

* [2018 Munich](http://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)

{% endblock %}

{% extends "layout.html" %}
{% block title %}ENGINEERING{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

## Overview

---

Our goal is to create a mutation-specific high-throughput glioma drug screening platform. To reach this goal, culminating in the development of NODES, we followed an iterative engineering design cycle:

![](https://static.igem.wiki/teams/4234/wiki/engfig.png)

Our engineering design cycle is driven by data from testing and stakeholder feedback to allow for effective iteration and prototyping.

## 1. Designing NODES

---

### Reporter Plasmids

While researching potential biosensors for D-2HG, we found a Nature Communications article by Xiao et al. outlining the discovery of an allosteric transcription factor, DhdR, from *Achromobacter denitrificans* NBRC 15125, which negatively regulates D-2HG dehydrogenase expression in response to D-2HG.
[[1]](https://doi.org/10.1038/s41467-021-27357-7) This article was the foundation of our reporter system. We ordered the human-codon optimized DhdR gene from Twist Bioscience, and inserted it into a commercially available pcDNA5/FRT plasmid (
[Thermo Fisher, V601020](https://www.thermofisher.com/order/catalog/product/V601020)) to serve as our biosensor.

Our reporter constructs consist of *dhdO* binding sites and a fluorescent (mCherry) reporter gene. Xiao et al. included a series of *dhdO* binding site sequences and their dissociation constants (KD), which reflect their binding affinities to DhdR
[[1]](https://doi.org/10.1038/s41467-021-27357-7). he authors tested 14 different binding site sequences, but our team decided to focus on the two with the lowest KD, *dhdO* 0# and *dhdO* 5# (Figure 1).

![](https://static.igem.wiki/teams/4234/wiki/engfig1.png)

Figure 1. Optimization of the D-2HG biosensor by mutating the cognate binding site. Figure 4a from “A D-2-hydroxyglutarate biosensor based on specific transcriptional regulator DhdR” [[1]](https://doi.org/10.1038/s41467-021-27357-7)

After choosing these two binding site sequences, we designed ten binding site combinations with varying numbers of repeats and spacer sequences to engineer cooperativity in the transcriptional response. Oligonucleotides containing binding site sequences were obtained from Integrated DNA Technologies. The motivation behind this was to optimize the binding kinetics of our system, with the hopes of creating a tunable reporter or introducing positive cooperative binding that enhances reporter activity. Thus, our team hopes to test the ten binding site combinations to determine which variation offers the largest dynamic range of expressions, as this would allow our system to effectively serve as a live cell biosensor for the compound of interest, D-2HG, based on intracellular concentrations (Figure 2).

![](https://static.igem.wiki/teams/4234/wiki/engfig2.png)

Figure 2. Graphic outlining the different binding site combinations that were synthesized for optimization

In a wild-type environment, without the presence of DhdR, we expect constitutive expression of the fluorescent protein as driven by CMV promoter expression. However, when DhdR is present, it will bind to the *dhdO* binding site, acting as a physical roadblock to transcription of our reporter gene. When D-2HG is elevated, as observed in *IDH1* mutant cells, it interacts with DhdR, releasing it from the *dhdO* binding site. This allows for transcription of the downstream reporter protein sequence, resulting in brighter expression that is visible in our in vitro system (Figure 3). Since D-2HG levels are elevated due to the *IDH1* mutation, we expect an increase in fluorescence due to the release of the DhdR caused by the binding of the upregulated oncometabolite. When we perform drug screening assays on our completed co-culture system, we will correlate decreased fluorescence with lower levels of D-2HG, corresponding to reduced tumor growth.

![](https://static.igem.wiki/teams/4234/wiki/engfig3.gif)

Figure 3. This figure outlines the mechanism of the interaction between the DhdR allosteric transcription factor and the *dhdO* binding site. The top of the figure outlines the normal expression of the reporter sequence, resulting in fluorescing cells. Case 1 is the wild-type condition with no D-2HG in the system. No fluorescence is expected in this case. Case 2 is the *IDH1* mutant condition when D-2HG is upregulated. We expect fluorescence expression because of the interaction between D-2HG and DhdR.

## 2. Building NODES

---

### Reporter Cloning

To develop a high-throughput, dynamically responsive drug screening platform, we needed to determine methods of characterizing cell behavior without lysing our samples. We first selected a reporter gene, mCherry, that has the ability to induce visually identifiable responses. We designed a plasmid that would constitutively express the desired mCherry fluorescence protein. To finalize the construction of the *dhdO* binding site constructs, we inserted ten variations of the binding site constructs using a restriction digest-ligation protocol into this constitutively expressing fluorescence plasmid, allowing the produced levels of mCherry to vary with the binding of the DhdR transcription factor (Figure 4).

![](https://static.igem.wiki/teams/4234/wiki/engfig5.png)

Figure 4: Plasmid maps of the DhdR constructs. Left: CMV with DhdR-NLS-FLAG. Right: CMV with FLAG-NLS-DhDR. Both inserts were added to the pcDNA5 backbone.

Based on our proposed system design, we then constructed a plasmid to express the allosteric transcriptional repressor DhdR. We have two versions of the construct with different nuclear localization sequences and FLAG tag placements (DhdR-NLS-FLAG vs. FLAG-NLS-DhdR) and cloned them into pcDNA5 plasmids to determine which sequence order allows for better protein expression (Figure 5). Since the DhdR protein is a transcription factor, it has to be localized to the nucleus after expression. Therefore, we added a nuclear localization sequence (NLS) to the construct. In addition, the FLAG tag was attached for ease of Western blotting to validate that the protein was being expressed in mammalian cells. We used the anti-FLAG M2 mouse antibody (
[Sigma Aldrich, F1804](https://www.sigmaaldrich.com/US/en/product/sigma/f1804) to detect our FLAG-tagged protein.

![](https://static.igem.wiki/teams/4234/wiki/engfig4.png)

Figure 5. Plasmid map of a Representative *dhdO* Binding Site Construct.

Additionally, we wanted to replicate the *IDH1* mutation in our co-culture system. To do so, we constructed two plasmids: one containing the wild type *IDH1* gene, and the other containing the *IDH1* R132 mutant gene
[[2]](https://doi.org/10.1038/s41388-017-0077-z) (Figure 6). Both variations of the gene were inserted into the pcDNA vector backbone.

![](https://static.igem.wiki/teams/4234/wiki/engfig6.png)

Figure 6: Plasmid map of the *IDH1* WT and *IDH1* R132 mutant constructs.

## 3. Testing NODES

---

### Validating the Amount of D-2HG Present in Various Cell Types

To test the basic expression level of D-2HG in the cell lines we house in the lab, we carried out a D-2HG concentration measurement for cell contents, culture supernatant, and blank media controls. We used the D-2HG assay kit for this measurement (
[Sigma Aldrich, MAK320-1KT](https://www.sigmaaldrich.com/US/en/product/sigma/mak320)). All our cell lines, organoids, supernatant, and blank media except for BXM4687 showed D-2HG levels below the detection range. HEK297 and A172 were both negative for intracellular D2HG, and were selected as recipients of the IDH plasmids to quantitatively study intracellular D-2HG production and reporter activity. BXM4687 will be subject to IDH1/2 locus sequencing to test for potential IDH mutations that lead to the accumulation of D-2HG.

### Testing the Reporter System with Dual Transfection and Dosing

To test the integration of the various parts of the proposed system, a D-2HG dosing assay was performed. HEK 293T cells were transfected with the *dhdO* binding site plasmid (specifically, construct #5 from Figure 2). In addition, they were transfected with either GFP plasmid (control) or DhdR-expressing plasmid (experimental), which allowed us to determine whether or not the presence of the DhdR-mCherry construct itself impacts the observed fluorescence levels. Finally, cells in both conditions were dosed with D-2HG at various concentrations (0 µM, 0.01 µM, 0.1 µM, 1 µM, 10 µM, 100 µM, 1000 µM), allowing us to determine how the amount of D-2HG present impacted the measured fluorescence. 48 hours after dosing, cell fluorescence was evaluated through flow cytometry (Figure 7).

![](https://static.igem.wiki/teams/4234/wiki/engfig7.png)

Figure 7: Graph showing the fluorescence levels under various conditions.

### Testing the Expression of DhdR in Mammalian Cells

Because the DhdR transcription factor was originally identified in a bacterial system, we wanted to validate that this protein would be expressed when translated to a mammalian system. To do this, we transfected the DhdR expression construct into HEK 293T cells and ran a Western blot on the cell lysate in order to validate the presence of the desired transcription factor (Figure 8). Because the DhdR sequence included in our plasmids contained a FLAG tag at the end, an anti-FLAG M2 mouse antibody (
[Sigma Aldrich, F1804](https://www.sigmaaldrich.com/US/en/product/sigma/f1804) was used for the identification of the protein of interest on the blot.

![](https://static.igem.wiki/teams/4234/wiki/engfig8.png)

Figure 8: Western blot results from SDS-PAGE of cell lysate, showing the presence of a band around 25 kDa in the 3’ DhdR and 5’ DhdR conditions and the lack of a band in the control condition

The presence of the band around 25 kDa indicated that the DhdR transcription factor of interest was being expressed in the transfected cells, validating that this component of our proposed system could be generated in mammalian cells. Further work would include running a native Western blot, which would allow us to validate that the DhdR transcription factor forms a dimer after expression in our mammalian system, as it does in the original bacterial system.

### Testing Primary Cell Transfection

Although transfections are widely used to transfer plasmid DNA into common cell lines such as HEK 293T cells, plasmid transfections of glioma cells have not been well studied. Limited synthetic biology work has been performed on primary cells, including glioma cell lines, and in organoid co-culture settings. From our lipofection and electroporation experiments,
**we concluded that the 250V condition had the best balance of transfection efficiency and fluorescence expression** (Figure 9).

![](https://static.igem.wiki/teams/4234/wiki/engfig9.png)

Figure 9. Electroporation of Patient-derived Glioma Cells using different voltage conditions (200V, 250V, and 300V) with FUGW-*GFP*

### Testing Tumor-Organoid Co-culture Drug Screening

To test and demonstrate the robustness of the co-culture system, we set up a drug assay experiment that mimicked the process of testing drugs with patient-derived cancer cell lines in a clinical setting. We used a patient-derived medulloblastoma cell line, D425-GFP, labeled with green fluorescent reporters. This line is known to be a fast-growing cancer cell line that is resistant to temozolomide (TMZ) at high doses. The drug assay using our co-culture system showed this trend as there were no significant differences in killing between the two TMZ doses (Figure 10).

![](https://static.igem.wiki/teams/4234/wiki/engfig10.png)

Figure 10. TMZ drug assay over the course of 14 hours. There were no significant differences between the two doses.

We tested another drug, medroxyprogesterone acetate (MPA), which has been confirmed to have potent killing effects on D425-GFP, to show that the co-culture system conferred the same results as in previous studies. The drug was verified in patient-derived xenograft (PDX) models and pre-established cell lines and has been shown to cause strong and fast killing of the D425 cell line. Our co-culture system confirmed this trend and had results that agreed with previously published data (Figure 11).

![](https://static.igem.wiki/teams/4234/wiki/engfig11.png)

Figure 11. The comparison between the killing effect of MPA over the course of 14 hours. Note that the two higher concentrations had no obvious differences in the killing.

The co-culture system also had the ability to demonstrate cell interactions and migrations visually. In a co-culture system, cancer cells migrate towards healthy cells, which is the target interaction some drugs inhibit. Upon introduction of cancer cells in the co-culture system, they tend to migrate towards healthy cells and invade the interior of healthy organoids. A drug that inhibits such an effect should then cause the cancer cells to remain dispersed randomly in the system, preventing the typical congregation of cancer cells at the edges of the organoids. We tested this idea in the co-culture system and it was clear that one verified drug, tazemetostat (EPZ), demonstrated this effect (Figure 12).

![](https://static.igem.wiki/teams/4234/wiki/engfig12.png)

Figure 12. Comparison of invasion distance between the cancer cells and the normal mini brains in untreated control and two different doses of EPZ groups. Note the different distances between the cancer cells and mini brains (arrows) increasing as the drug concentration increases, and the different total amount of cancer cells (green) in each condition.

This demonstrated the functionality of our current co-culture platform, as it accurately recapitulated the differences between drug effects in varying doses and visually provided evidence that indicates inhibition of cancer cell migration. From this drug assay, we conclude that there is a high likelihood that our co-culture system holds accurate predictive power in regard to forecasting drug responses in vivo, and has a potential application in clinical settings.

## 4. Refining NODES

---

Despite being the most common malignant brain tumor, clinical treatment of glioma is severely limited by the lack of a scalable, physiologically-relevant model for testing therapeutics. To fully understand the implications of this problem, our team talked with researchers and individuals who work directly with patients to understand how our project scope should be refined to best address the needs of key stakeholders.

Throughout our engineering design process, we integrated stakeholder feedback to guide the development of our drug screening platform. Based on what we learned from the interviews, we verified the need for a glioma drug screening platform, pivoted to a preclinical trial drug screening tool, and shifted to a mutation-specific reporter system. To learn more about how we integrated stakeholder feedback to refine our project scope and execution, see the
[Human Practices](https://2022.igem.wiki/duke/human-practices) page.

![](https://static.igem.wiki/teams/4234/wiki/engfig13.png)

Figure 13. Refining project scope and execution

## References

---

- Xiao, D., Zhang, W., Guo, X., Liu, Y., Hu, C., Guo, S., Kang, Z., Xu, X., Ma, C., Gao, C., & Xu, P. (2021). A D-2-hydroxyglutarate biosensor based on specific transcriptional regulator DHDR. Nature Communications, 12(1). https://doi.org/10.1038/s41467-021-27357-7
- Molenaar, R. J., Maciejewski, J. P., Wilmink, J. W., & van Noorden, C. J. (2018). Wild-type and mutated IDH1/2 enzymes and therapy responses. Oncogene, 37(15), 1949–1960. https://doi.org/10.1038/s41388-017-0077-z

{% endblock %}

{% extends "layout.html" %}
{% block title %}EXPERIMENTS{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## Wet Lab Protocols

### DNA Sourcing

The templates and sources for the various PCR products are listed below:

Table 1: PCR Products

| **PCR Product Used in Gibson** | **Template Source Plasmid** | **Source Company/Notes** |
| --- | --- | --- |
| pcDNA5 backbone | pcDNA5\_FRT\_mCH | Addgene, #127109 |
| mCherry | pcDNA5\_FRT\_mCH | Addgene, #127109 |
| CMV promoter | pcDNA5-FRT-mCH | Addgene, #127019 |
| DhdR-NLS-FLAG gene, FLAG-NLS-DhdR gene | Directly synthesized gene block | Twist Biosciences |

PCR primers were purchased from IDT and the sequences used are shown here. The DNA constructs used for the binding sites were purchased as single strands from IDT and annealed in the lab.

### Gibson Assembly Cloning

The constitutive reporters and DhdR constructs were produced using Gibson Assembly. The workflow for plasmid generation is shown below:

![](https://static.igem.wiki/teams/4234/wiki/pcr-cloning-workflow.png)

Figure 1. PCR/Cloning Workflow

**[PCR](https://drive.google.com/open?id=1a3SSPUps8ETEwseP8IAaowglLucH5VFE)**
This was performed using Q5 master mix (NEB, cat. #M0491S).

**[Gel Electrophoresis](https://drive.google.com/open?id=1AJvuEsLhtYftmevxIUzooD6T4MzViHPX)**

**[DpnI Digest](https://drive.google.com/file/d/1NhuEWDo0glXuR9HNuMIRWCKqFRw5N55k/view?usp=sharing)**
This was performed using Dpn1 enzyme (NEB, cat. #R0176S).

**[PCR Purification](https://drive.google.com/file/d/1Pt0LcGi5OLScN9MiJP5ioFkY2RATLWhY/view?usp=sharing)**
This was performed using Monarch PCR & DNA Cleanup Kit (NEB, cat #T1030S) as well as DNA Clean and Concentrator (Zymo Biosciences, cat. #D4013).

**[Gel Extraction](https://drive.google.com/file/d/1uZQq_3SAn3Gi8wDNqQLXqnfTIGaCMOCH/view?usp=sharing)**
This was performed using Zymoclean Gel DNA Recovery Kit (Zymo Biosciences, cat. #D4007).

**[Gibson Assembly](https://drive.google.com/file/d/1ZDbXg6rEJysYiUouF2Lf3QCZauUugW-W/view?usp=sharing)**
This was performed using homemade Gibson master mix, which included DNA polymerase, DNA ligase, and 5’ endonuclease in buffer solution

### Restriction Enzyme Cloning

The binding site plasmids, both in pcDNA5 and constitutive reporter, were produced using restriction enzyme cloning.

**[Oligonucleotide Annealing](https://drive.google.com/open?id=1tQZafyjt17RQWmvZUTE6aLLYEbyQDLII)**
This was performed using T4 DNA Ligase Reaction Buffer (NEB, cat. #B0202S) and T4 PNK (NEB, cat. #M0201S)

**[Backbone Digest](https://drive.google.com/file/d/1gvc_kirOUSbjSgGNtc-nF0DuCU5i6Tcj/view?usp=sharing)**
This was performed using the restriction enzymes NheI HF (NEB, cat. #R3131S) and AflIII (NEB, cat. #R0541S). Additionally, the reaction was carried out in rCutSmart buffer (NEB, cat. #B6004S).

**[Ligation](https://drive.google.com/open?id=1KKf7QTLuOXR2AtNmgjUdAq4kHw77gMwY)**
This was performed using Quick Ligase (NEB, cat. #M2200S) in Quickligation Buffer (NEB, cat. #B6058S).

### Bacterial Work

**[Bacterial Transformation](https://drive.google.com/file/d/1No1QmiSoRQHuNQlh5-IUj3Lz9PZvvKLa/view?usp=sharing)**
For transformations, we utilized Stable 3 cells (NEB, C3040H) and 5-alpha cells (NEB, C2988J). Agar plates were made with carbenicillin and homemade LB agar.

**[Bacterial Streaking](https://drive.google.com/file/d/1-Junf0SzAbwu1ISekrLuBYKccUMIqwRL/view?usp=sharing)**

**[Bacterial Inoculations](https://drive.google.com/file/d/1MPZyq4kKyrxe01y69Wl0KE2fwnsOoe-F/view?usp=sharing)**
For inoculations, the media used was liquid LB broth with carbenicillin.

**[Miniprep](https://drive.google.com/file/d/1-01LD0ba7ivXSFTz1Be_RjfLu_rdywPc/view?usp=sharing)**
For minipreps, we utilized several different kits, including Zyppy plasmid miniprep kit (Zymo Biosciences, D4036), Zymo classic miniprep kit (Zymo Biosciences, D4054), and Monarch Plasmid Miniprep kit (NEB, T1010S).

### Cell Work

**[Cell Culture](https://drive.google.com/open?id=1Vi2QSl52If8v-YGc1Duw3NxY4bmQcJ2E)**
This protocol outlines the maintenance of our three main cell lines, as well as cell counting and fixing. For passaging HEK and BXM cells,
we utilized media made with DMEM (Gibco, 1193065), FBS (free sample provided by Gibco), and alpha-alpha (Gibco). Cell detachment required TrypLE
(Gibco, 12605010) and PBS (Gibco, 10010002). Cell counting was performed with trypan blue (Gibco, 15250061).

**[Transfection](https://drive.google.com/open?id=1y5LhMLLkGVLBvMsvIWSjNnUJInYp1hT5)**
For lipofections, two different forms of reagent were used. For standard HEK transfections, we used Lipofectamine 2000 (Thermo, 11668019) and Optimem
(Gibco, 31985070). For non-standard BXM transfections, we used Lipofectamine LTX with PLUS reagent (Thermo, 15338100) and Optimem (Gibco, 31985070).

**[Luciferase Assay](https://drive.google.com/drive/folders/12iHE53T5bKEGZIH8FnU9nrHt3hbCaEiP)**
For this assay, the Pierce Cypridina Luciferase Glow Assay Kit (Thermo Fisher, 16170) was used.

### Western Blot

**[iBlind Flex Western Protocol](https://drive.google.com/file/d/1XTJnfCGCRAH14_gHBwpvgMvrWcH9UwNb/view?usp=sharing)**

**[Power Blotter Protocol](https://drive.google.com/file/d/1-SxOzxPT57GJbLlCZO9wTtag8wOvpei1/view?usp=sharing)**

**[MilliporeSigma Protocol](https://drive.google.com/file/d/1of7nj_GeMMsEZcJ9FmehXP4rh7_8IVB3/view?usp=sharing)**

### Flow Cytometry

**[Collecting Cells](https://drive.google.com/file/d/14P850jUZH5ONrnIMsqxMLNE8KY_YcZkX/view?usp=sharing)**

**[Data Analysis](https://drive.google.com/file/d/1GAlVwuuvBjrqqkslUsrRFYtv0sRKSoxE/view?usp=sharing)**

## Stem Cell Protocol

### hiPSC Culture

Green fluorescent protein (GFP) labeled human induced pluripotent stem cells (hiPSC) wereobtained at passage 2. The iPSCs were seeded onto pre-coated six-well plates using
iPSC Coating Solution (Angio-Proteomie, cat. No. cAP-50). Media change was done daily with human iPSC culture medium (Xeno-free, serum-free, stable; Angio-Proteomie, cat. No.
cAP-49). The cells were passaged at around 80% or higher confluency. Essentially, media was removed from the plate and 1 mL iPSC non-enzymatic dissociation solution (Angio-Proteomie,
cAP-51) was added. The plate was then incubated at 37 °C for 5 minutes and the colonies were collected by pipetting up and down 3 times. The dissociation solution was removed with
centrifugation at 200 g for 5 minutes, and iPSCs were seeded into pre-warmed iPSC culture medium with 10 µM ROCK inhibitor (Y-27632, Selleckchem-S1049). The iPSC cultures were
incubated at 37 °C in 5% CO2. iPSC quality was examined using the Human Pluripotent Stem Cell Naive State qPCR Array (STEMCELL cat. No.07523) and was determined to be at prime state for differentiation.

### Immunofluorescent Staining

Organoids were fixed in 4% paraformaldehyde overnight at 4 °C and allowed to sink in 30% sucrose overnight, followed by rapid freezing in Tissue-tek OCT Compound (Sakura Finetek #4583). Organoids were
cryosectioned with a thickness of 10 µm. To stain, the sections were blocked in 0.25% Triton X-100 in TBS at 4 °C overnight. Primary antibodies were incubated with the sections with 10% horse serum and
1% BSA overnight at 4 °C, followed by secondary antibody incubation at the same conditions. The primary antibodies were diluted with the following dilutions: TUJ1 (BioLegend MMS-435P, 1:750), FOXG1
(Abcam ab196868, 1:200), PAX6 (DSHB, 1:200), FZD9 (OriGene TA344067, 1:200), GBX2 (Santa Cruz sc-81963, 1:200), PAX2 (Abnova M01, 1:200), SOX2 (Chemicon, AB5603, 1:300). Secondary antibodies were donkey
Alexa Fluor 488 and 594. Nucleus was stained with DAPI.

### Establishing Glioma and Mini Brain Co-Culture System for Drug Screening

For the co-culture system, we propose a general protocol here for future co-culture experiments:

1. Briefly, a 96-well ultra-low attachment U-bottom plate is used to set up the experiment.
2. The plate should include positive and negative control wells, i.e. mini brain only and glioma cell only conditions, with at least 3 replicates for each to avoid within group variations.
3. Use cerebral organoid media in the co-culture system to ensure best performance.
4. A low density of glioma cells should be seeded into each well. We recommend the amount of 500 cells/well in consideration of the small volume of the well. If the cancer cell line is fast-growing or drug-resistant, decrease seeding density.
5. Add an equal amount of mini brains in each well. As a U-bottom plate is used, the mini brains will automatically collect to the bottom of the well and should be ideal for imaging purposes.
6. Observe the plate over the course of 3-5 days. Take images regularly to document invasion behavior.

Downstream: The plate can be dosed with different drugs for screening purposes:

1. We recommend having at least 3 titrations of concentration for each drug tested to observe both the killing effect and the potential toxicity of the drug on normal brain cells.
2. The drug and the glioma cells should be dosed at the same time on day 0 into the co-culture system.
3. Set up control wells where mini brains are dosed with glioma cells but no drug. They monitor the invasiveness of the glioma cells, and if no invasion is observed, the other wells stand no significant meaning.
4. Observe over the course of 3-5 days and take images regularly to document the number and status of cells, migrations, and invasion.

## Safe Lab Practices

Given the hazardous nature of some of the organisms and reagents used in synthetic biology research, the Duke iGEM team closely followed general lab safety protocols:

* Safety training and certification through Duke OESO which included general lab safety training, BSL-2 training, and other relevant topics
* Upkeep and maintenance of relevant safety equipment in the lab, including safety showers and eye wash stations
* Requiring that all food and water is left outside of the lab
* Safely disposing of hazardous materials, like Sybr Safe or broken glass, into appropriate containers
* Proper usage of personal protective equipment such as gloves and lab coats at all times.
* Maintaining an open line of communication with advisors and lab managers regarding any concerns with safe lab practice.

Additionally, because our work involved management of E. coli and human stem cells, we made sure to follow safety procedures relevant to biosafety level 2 environments:

* Handling biological samples using the Class A2 laminar flow biosafety cabinet
* Using commercially available E. coli strains that have been selected for low pathogenicity
* Adhering to proper waste disposal protocols ensuring that all materials utilized in bacterial or cellular culture work were properly bleached or autoclaved prior to regular trash disposal
* Storing cell culture flasks within a separately designated cell incubator marked clearly with biohazard indications

## Safety and Security of Implementation

Ultimately we hope that our reporter system can be used by other researchers and implemented as an effective drug screening tool in
clinical research environments for real patients suffering from gliomas and other cancers. With the hope of implementing our device
outside of a basic laboratory environment, we had to consider what safety concerns our system would pose to the real world.

We envision that our products will be primarily used in a clinical research setting, thus presenting an extremely low risk of being
introduced into the environment. We acknowledge, however, that there will be a possibility that the final product we deliver will be
assessed to pose a potential threat when in contact with the outside environment, and a kill-switch will be implemented on the system
upon need. Our guiding principle is that we will limit our products from contacting the environment as much as possible, and if contact
is necessary, an intrinsic kill-switch will be installed to prevent escape of recombinant DNA.

{% endblock %}

{% extends "layout.html" %}
{% block title %}RESULTS{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

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

## Describe what your results mean

---

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

## Project Achievements

---

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

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

## Inspirations

---

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

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