iGEM 2021 — Team Duke

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

# Team:Duke/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

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

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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

# Team:Duke/Engineering

![](https://static.igem.org/mediawiki/2021/8/81/T--Duke--img--igem_montserrat.png)

* Vision

  [Description](https://2021.igem.org/Team%3ADuke/Description)
  [Design](https://2021.igem.org/Team%3ADuke/Design)
* Foundation

  [Minibrain](https://2021.igem.org/Team%3ADuke/Results_Minibrain)
  [Cloning](https://2021.igem.org/Team%3ADuke/Results_Cloning)
  [Parts](https://2021.igem.org/Team%3ADuke/Parts)
  [Experiments](https://2021.igem.org/Team%3ADuke/Experiments)
* Product

  [Proof of
  Concept](https://2021.igem.org/Team%3ADuke/Proof_Of_Concept)
  [Model](https://2021.igem.org/Team%3ADuke/Model)
  [Software](https://2021.igem.org/Team%3ADuke/Software)
  [Implementation](https://2021.igem.org/Team%3ADuke/Implementation)
* Human
  Practice

  [Integrated
  Human
  Practice](https://2021.igem.org/Team%3ADuke/Human_Practices)
  [Communication
  and
  Education](https://2021.igem.org/Team%3ADuke/Communication)
  [Collaboration](https://2021.igem.org/Team%3ADuke/Collaboration)
  [Partnership](https://2021.igem.org/Team%3ADuke/Partnership)
* [Safety](https://2021.igem.org/Team%3ADuke/Safety)
* Team

  [About
  Us](https://2021.igem.org/Team%3ADuke/About_Us)
  [Attributions](https://2021.igem.org/Team%3ADuke/Attributions)
  [Engineering](https://2021.igem.org/Team%3ADuke/Engineering)
  [Contribution](https://2021.igem.org/Team%3ADuke/Contribution)

# 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.org/mediawiki/2021/1/1c/T--Duke--img--engineering--image10.png)

Our engineering design cycle is driven by data from testing and stakeholder feedback
to allow
for effective iteration and prototyping. Key sections from the [Design](https://www.google.com/url?q=https://2021.igem.org/Team:Duke/Design&sa=D&source=editors&ust=1634873910782000&usg=AOvVaw2LARbjSUC89pd1pxApN92L), Results
([Cloning](https://www.google.com/url?q=https://2021.igem.org/Team:Duke/Cloning&sa=D&source=editors&ust=1634873910783000&usg=AOvVaw0xZfQfq83UGpET_wV-zogC),
[Minibrain](https://www.google.com/url?q=https://2021.igem.org/Team:Duke/Minibrain&sa=D&source=editors&ust=1634873910783000&usg=AOvVaw3Izau4puUc_yniF_nwSIfv)), [Proof
of Concept](https://www.google.com/url?q=https://2021.igem.org/Team:Duke/Proof_Of_Concept&sa=D&source=editors&ust=1634873910784000&usg=AOvVaw30VCAqVCs3GlrRDXmR28du), and [Human
Practice](https://www.google.com/url?q=https://2021.igem.org/Team:Duke/Human_Practices&sa=D&source=editors&ust=1634873910784000&usg=AOvVaw2K5KUh6HqGjR2FvDa_yTAi) pages are included in this page to
showcase how we integrated all aspects of our work within
an
engineering design process.

# 1. Designing NODES

## Reporter Plasmids

While researching potential biosensors for D-2-HG, we found a Biorxiv article by
Xiao et al.
outlining the discovery of an allosteric transcription factor, DhdR, from Achromobacter
denitrificans NBRC
15125, which negatively regulates D-2-HG dehydrogenase expression in response to D-2-HG. 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 plasmid (Thermo Fischer, V103320) to serve as our
biosensor.

Our reporter constructs consist of dhdO binding sites
and a fluorescent (tdTomato) or luminescent (CLuc) reporter gene. Xiao et al. included a series
of
dhdO binding sites sequences and their KD dissociation
constants, which
reflect their binding affinities to DhdR. The 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). After choosing these two
binding site
sequences, we designed ten binding site combinations with varying numbers of repeats and spacer
sequences,
which were all obtained from Integrated DNA Technology. The motivation behind this was to
optimize the
binding of our system, with the hopes of creating a tunable reporter or a cooperative binding
that enhances
reporter activity. The presence of binding sites and variations in the number of repeats can
impact the
binding behavior of associated proteins. 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 biosensor for the compound of interest, D-2-HG.

![](https://static.igem.org/mediawiki/2021/6/60/T--Duke--img--engineering--image6.png)

##### Figure 1. Optimization of the D-2-HG biosensor from “A D-2-hydroxyglutarate biosensor based on specific transcriptional regulator DhdR” (Xiao et al., 2021)

In a wild-type environment, without the
presence of DhdR,
we expect normal expression of the fluorescent or luminescent protein. However, when
DhdR is present, it will bind to the dhdO
binding site, allosterically blocking the transcription of our reporter gene. When
D-2-HG is
elevated, as observed in IDH1 mutant cells, it binds to
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 vivo droplet system (Figure
2). Since
D-2-HG levels are elevated due to the IDH1 mutation, we
expect an increase in fluorescence or luminescence 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 associate decreased fluorescence or luminescence with lower levels of D-2-HG, corresponding
to reduced
tumor growth.

![](https://static.igem.org/mediawiki/2021/f/f7/T--Duke--img--engineering--image11.png)

##### Figure 2. 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 normal expression of the reporter sequence, resulting in fluorescing cells. Case 1 is the wildtype condition with no D-2-HG in the system. No fluorescence is expected in this case. Case 2 is the IDH1 mutant condition, when D-2-HG is upregulated. We expect fluorescence expression because of the interaction between D-2-HG and DhdR.

For more information, see the [Design](https://www.google.com/url?q=https://2021.igem.org/Team:Duke/Design&sa=D&source=editors&ust=1634873910786000&usg=AOvVaw1gnwKxsS_r1uWILph-eP_v) page.

## High-throughput Organoids

Microfluidics is an emerging field of technology that enhances the
ability to handle
very small volumes of fluid in an automatic and high-throughput manner. We capitalized on this
new
technology by applying it to the early stages of organoid development, which allowed us to
overcome any
previous barriers that hindered the mass generation of embryoid bodies. By using microfluidics
to produce
organoids encapsulated in droplets, we are able to improve upon traditional methods of
generating cerebral
organoids. These droplets significantly enhance the minibrain production rate, allowing for
larger batch
sizes and lower overall maintenance. This enables the development of high-throughput drug
screening
platforms that can test a multitude of drugs with the same cohort of cerebral organoids
co-cultured with
patient-derived glioma cell lines to reduce batch variability. Further, since the current
cerebral organoid
differentiation process heavily depends on the self-patterning of cell clusters, applying
microfluidics
technology can ensure consistency within one batch of minibrains by generating droplets
containing small
cell aggregates of a certain cell number. With the abovementioned advantages of the
microfluidics system,
this technology was incorporated into our design to generate a high-throughput organoid
production pipeline
(Figure 3).

![](https://static.igem.org/mediawiki/2021/a/ac/T--Duke--img--engineering--image1.png)

##### Figure 3. The timeline of the droplet-based mini brain workflow

# 2. Building NODES

## Reporter Cloning

To develop a high-throughput drug screening platform, we needed to determine methods
of
characterizing cell behavior without lysing our samples. We first selected reporter genes
(tdTomato and CLuc) that encode fluorescent and luminescent
proteins, respectively,
that have the ability to induce visually identifiable responses. To establish the backbones for
creating our
overall reporter system constructs, the team designed four plasmids consisting of different
promoter (CMV
and hUBC) and reporter gene (tdTomato and CLuc) combinations (Figure 4).

![](https://static.igem.org/mediawiki/2021/d/d9/T--Duke--img--engineering--image13.png)

##### Figure 4. Plasmid maps of the Constitutive Reporter constructs. Top Left: CMV with tdTomato. Top Right: CMW with CLuc. Bottom Left: hUBC with tdTomato. Bottom right: hUBC with CLuc

To construct the second component of our reporter system (the
DhdR binding site constructs), we inserted ten variations
of the binding site constructs into a
commercially available pcDNA5 plasmid, which encodes for the red fluorescent protein mCherry.
Based on our
proposed system design, we then constructed a plasmid to express the allosteric transcription
repressor
DhdR. We have two versions of the construct with different nuclear localization sequence and
FLAG tag
placements (DhdR-NLS-FLAG vs. FLAG-NLS-DhdR) and will clone them into pcDNA5 plasmids to
determine which
sequence order allows for better protein expression (Figure 5).

![](https://static.igem.org/mediawiki/2021/d/d5/T--Duke--img--engineering--image14.png)

##### Figure 5: Plasmid maps of the DhdR constructs. Left: CMV with DhdR-NLS-FLAG. Right: CMW with FLAG-NLS-DhDR.

For more details, see the [Results](https://www.google.com/url?q=https://2021.igem.org/Team:Duke/Cloning&sa=D&source=editors&ust=1634873910789000&usg=AOvVaw1cpkvvhkNZtmRzcYTi86ry) page.

## High-throughput organoid platform

Current methods for generating cerebral organoids are labor-intensive and extremely
time
consuming, mainly due to the individualized handling of each of the mini brains in various steps
such as
transferring and encapsulating in extracellular matrix (ECM) dorms (for more
details, see
[Design](https://www.google.com/url?q=https://2021.igem.org/Team:Duke/Design&sa=D&source=editors&ust=1634873910789000&usg=AOvVaw37-GxEjVLxk-0ZIvIjkIKB) page. We refined the existing protocol for cultivating cerebral organoids (mini brains)
to enhance
its through-put and reduce the human labor needed by using the microfluidics system. Briefly, we
incorporated microfluidics technology on day 0 to make hiPSC droplets that directly packaged a
certain
amount of stem cells into a Matrigel sphere. By pre-packaging the stem cells into Matrigel
droplets, we were
able to generate hundreds to thousands of embryoid bodies in one cohort and reduced much time
and labor
needed later in the process by the original protocol. The mini brains generated from the
droplet-based
method recapitulated all the crucial stages in cerebral organoid differentiation process
including embryoid
bodies, neuroectoderm- and neuroepithelium-like structures, and cerebral tissues. The mature
organoids
exhibited complex, heterogeneous tissue characteristics, and can be kept alive indefinitely
under proper
maintenance. From here, we are confident with the quality of our mini brains in regard to
mimicking human
brain microenvironment, and therefore will move onto testing the integration of our engineering
products.

# 3. Testing NODES

## Testing Reporter Circuit

To verify the fluorescence gene expression of tdTomato, we
transfected huBC-tdTomato into HEK293T cells, a widely
used cell
line, for initial validation of our construct. The cells were successfully transfected with
moderate
efficiency (~40%) and demonstrated fluorescence expression. The successful
fluorescence
expression of huBC-tdTomato in HEK293T cells validates
that our
construct can be used as an imaging marker for synthetic biology applications to characterize
different cell
behaviors, such as changes in D-2-HG (Figure 6).

![](https://static.igem.org/mediawiki/2021/3/34/T--Duke--img--engineering--image2.png)

##### Figure 6. Fluorescent microscopy verification of the expression of the huBC-tdTomato construct  in HEK293T cells.

To verify the CMV-CLuc and huBC-CLuc constructs, we
transfected
them into HEK293T cells and performed a luciferin-based luminescence assay. We added vargulin, a
luciferin
that releases light in the presence of a luciferase, to HEK 293T cells transfected with various
amount of
CMV-CLuc and hUBC-CLuc DNA (0 ng, 100 ng, 250 ng, and 500 ng). The HEK293T cells with no
transfections had
no fluorescence intensity. The fluorescence intensity was significantly greater in cells with
CMV-CLuc
(Figure 7, left) and huBC-CLuc (Figure 7, right) transfected
compared to
non-transfected cells, verifying that the two CLuc constitutive reporters we designed secreted
luciferase
enzymes.

![](https://static.igem.org/mediawiki/2021/1/16/T--Duke--img--engineering--image12.png)![](https://static.igem.org/mediawiki/2021/0/0b/T--Duke--img--engineering--image5.png)

##### Figure 7. Luciferase Assay of CMV-CLuc (left) and huBC-CLuc (right) in HEK293T cells with various DNA transfection amounts (0 ng, 100 ng, 250 ng, and 500 ng)

For more details of our verification process, see the [Results](https://www.google.com/url?q=https://2021.igem.org/Team:Duke/Cloning&sa=D&source=editors&ust=1634873910791000&usg=AOvVaw1Q6DYeXO6t2ex0_l3zBt6E) page.

## 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
8). For more details of our transfection testing process, see the [Proof
of Concept](https://www.google.com/url?q=https://2021.igem.org/Team:Duke/Proof_Of_Concept&sa=D&source=editors&ust=1634873910792000&usg=AOvVaw3M-dy56KNR-1lNnnh7L15N) page.

![](https://static.igem.org/mediawiki/2021/5/51/T--Duke--img--engineering--image7.png)

##### Figure 8. Electroporation of Patient-derived Glioma Cells using different voltage conditions (200V, 250V, and 300V) with FUGW-GFP

## Comparing High-throughput Organoids With Conventional Organoids

We established a comparison between the conventional and droplet-based
methods to
demonstrate the superiority of our droplet-based method. For the conventional method, we
strictly followed
the protocol accompanying commercially available cerebral organoid cultivation kits, and had a
droplet-based
group that was started on the same day as a comparison counterpart. Equal amounts of cells were
harvested
for the generation of embryoid bodies on day 0. Morphologically, the mini brains generated from
droplets
versus conventional methods displayed similar tissue development timeline and patterns while
conferring a
high throughput. Further, we noticed that there was a large percentage lost with the
conventional method due
to embryoid body/organoid fusion and handling errors that damaged embryoid bodies. Out of the 40
embryoid
bodies we seeded, only around 10 individual mini brains were able to mature and develop. The
droplet method,
although seeing some fusion events too, had more embryoid bodies to start with and therefore
yielded more
mini brains. Additionally, we observed that the droplets fused into large aggregates in each
well after an
extensive period of time (2 weeks) of the orbital shaker. We therefore argue that the usage of
an orbital
shaker or a bioreactor would not be required for our droplet-based mini brains. In sum, our
droplet-based
method for generating mini brains demonstrated obvious advantages over the conventional method,
and provided
a foundational part to the establishment of the co-culture system.

## 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 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 in two different TMZ doses (Figure 9).

![](https://static.igem.org/mediawiki/2021/d/d5/T--Duke--img--engineering--image4.png)

##### Figure 9. 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 10).

![](https://static.igem.org/mediawiki/2021/7/73/T--Duke--img--engineering--image8.png)

##### Figure 10. 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 killing.

The co-culture system also had the ability to demonstrate cell
interactions and
migrations visually. Cancer cell lines are usually known to be attracted to healthy cells in a
co-culture
system, 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 11).

##### ![](https://static.igem.org/mediawiki/2021/1/1b/T--Duke--img--engineering--image3.png) Figure 11. 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 have 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://www.google.com/url?q=https://2021.igem.org/Team:Duke/Human_Practices&sa=D&source=editors&ust=1634873910795000&usg=AOvVaw008ZErQ_ezuPns9YZaMi6N) page.

### ![](https://static.igem.org/mediawiki/2021/e/ec/T--Duke--img--engineering--image9.png)

##### Figure 12. 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., &amp; Xu, P. (2021). A D-2-hydroxyglutarate biosensor based on specific
transcriptional
regulator DHDR. https://doi.org/10.1101/2021.02.18.430539

Duke iGEM © 2021

Duke iGEM © 2021

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

# Team:Duke/Experiments

[![](https://static.igem.org/mediawiki/2021/8/81/T--Duke--img--igem_montserrat.png)](https://2021.igem.org/Team%3ADuke)

* Vision

  [Description](https://2021.igem.org/Team%3ADuke/Description)
  [Design](https://2021.igem.org/Team%3ADuke/Design)
* Foundation

  [Minibrain](https://2021.igem.org/Team%3ADuke/Results_Minibrain)
  [Cloning](https://2021.igem.org/Team%3ADuke/Results_Cloning)
  [Parts](https://2021.igem.org/Team%3ADuke/Parts)
  [Experiments](https://2021.igem.org/Team%3ADuke/Experiments)
* Product

  [Proof of
  Concept](https://2021.igem.org/Team%3ADuke/Proof_Of_Concept)
  [Model](https://2021.igem.org/Team%3ADuke/Model)
  [Software](https://2021.igem.org/Team%3ADuke/Software)
  [Implementation](https://2021.igem.org/Team%3ADuke/Implementation)
* Human
  Practice

  [Integrated
  Human
  Practice](https://2021.igem.org/Team%3ADuke/Human_Practices)
  [Communication
  and
  Education](https://2021.igem.org/Team%3ADuke/Communication)
  [Collaboration](https://2021.igem.org/Team%3ADuke/Collaboration)
  [Partnership](https://2021.igem.org/Team%3ADuke/Partnership)
* [Safety](https://2021.igem.org/Team%3ADuke/Safety)
* Team

  [About
  Us](https://2021.igem.org/Team%3ADuke/About_Us)
  [Attributions](https://2021.igem.org/Team%3ADuke/Attributions)
  [Engineering](https://2021.igem.org/Team%3ADuke/Engineering)
  [Contribution](https://2021.igem.org/Team%3ADuke/Contribution)

# Wet Lab Protocols

## DNA sourcing

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

|  |  |  |
| --- | --- | --- |
| PCR Product Used in Gibson | Template Source Plasmid | Source Company/Notes |
| pcDNA5 backbone | pcDNA5-FRT-mCh | Addgene, #127109 |
| hUBC promoter | FUGW-hUBC | Gersbach Lab, Duke |
| CMV promoter | pcDNA5-FRT-mCh | Addgene, #127019 |
| cLuc gene | pIND-cLuc | Addgene, #53224 |
| tdTomato gene | pCRISPR\_HOT-tdTomato | Addgene, #138567 |
| 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](https://www.google.com/url?q=https://docs.google.com/spreadsheets/d/10hvRYkmJbzIvGL1Uz1LWNUungyksDnQmw2k255sovfs/edit?usp%3Dsharing&sa=D&source=editors&ust=1634875622026000&usg=AOvVaw0juYFFSeXztxr9nXB7yXG9). The DNA constructs used for the binding sites were purchased as single strands
from IDT and
annealed in 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.org/mediawiki/2021/b/b5/T--Duke--img--experiments--image1.png)

[PCR](https://www.google.com/url?q=https://drive.google.com/open?id%3D1a3SSPUps8ETEwseP8IAaowglLucH5VFE&sa=D&source=editors&ust=1634875622027000&usg=AOvVaw1ux3bd4uzrvgR7PMYsyTyT)

This was performed using Q5 master mix (NEB, cat. #M0491S).

[Gel
Electrophoresis](https://www.google.com/url?q=https://drive.google.com/open?id%3D1AJvuEsLhtYftmevxIUzooD6T4MzViHPX&sa=D&source=editors&ust=1634875622028000&usg=AOvVaw2WBnMIITFwPimy60U8DHFi)

[DpnI
Digest](https://www.google.com/url?q=https://drive.google.com/file/d/1NhuEWDo0glXuR9HNuMIRWCKqFRw5N55k/view?usp%3Dsharing&sa=D&source=editors&ust=1634875622029000&usg=AOvVaw2jvX7RH5TM3g-ZrVOVtLC2)

This was performed using Dpn1 enzyme (NEB, cat. #R0176S).

[PCR
Purification](https://www.google.com/url?q=https://drive.google.com/file/d/1Pt0LcGi5OLScN9MiJP5ioFkY2RATLWhY/view?usp%3Dsharing&sa=D&source=editors&ust=1634875622029000&usg=AOvVaw3yg2TnZhUQFjYpVqv-Tqve)

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://www.google.com/url?q=https://drive.google.com/file/d/1uZQq_3SAn3Gi8wDNqQLXqnfTIGaCMOCH/view?usp%3Dsharing&sa=D&source=editors&ust=1634875622030000&usg=AOvVaw2mtEiVteQLWmoNd7pfmKSX)

This was performed using Zymoclean Gel DNA Recovery Kit (Zymo
Biosciences, cat.
#D4007).

[Gibson
Assembly](https://www.google.com/url?q=https://drive.google.com/file/d/1ZDbXg6rEJysYiUouF2Lf3QCZauUugW-W/view?usp%3Dsharing&sa=D&source=editors&ust=1634875622031000&usg=AOvVaw1qkj048VaJv1YKIOJDeFog)

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://www.google.com/url?q=https://drive.google.com/open?id%3D1tQZafyjt17RQWmvZUTE6aLLYEbyQDLII&sa=D&source=editors&ust=1634875622032000&usg=AOvVaw3f2Gzz4k7Zr420EI3cFeQN)

This was performed using T4 DNA Ligase Reaction Buffer (NEB, cat.
#B0202S) and T4
PNK
(NEB, cat. #M0201S).

[Backbone
Digest](https://www.google.com/url?q=https://drive.google.com/file/d/1gvc_kirOUSbjSgGNtc-nF0DuCU5i6Tcj/view?usp%3Dsharing&sa=D&source=editors&ust=1634875622033000&usg=AOvVaw0dVgCZuNtU9LbUDIy0AkJ1)

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://www.google.com/url?q=https://drive.google.com/open?id%3D1KKf7QTLuOXR2AtNmgjUdAq4kHw77gMwY&sa=D&source=editors&ust=1634875622034000&usg=AOvVaw3FdLXmyt-uXOaauGIBuZc7)

This was performed using Quick Ligase (NEB, cat. #M2200S) in
Quickligation Buffer
(NEB, cat. #B6058S).

## Bacterial Work

[Bacterial
Transformation](https://www.google.com/url?q=https://drive.google.com/file/d/1No1QmiSoRQHuNQlh5-IUj3Lz9PZvvKLa/view?usp%3Dsharing&sa=D&source=editors&ust=1634875622035000&usg=AOvVaw3yFcQi2AzuskLtXjK08h14)

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://www.google.com/url?q=https://drive.google.com/file/d/1-Junf0SzAbwu1ISekrLuBYKccUMIqwRL/view?usp%3Dsharing&sa=D&source=editors&ust=1634875622036000&usg=AOvVaw1RhKwe-LaHJiyFUR1FRWeC)

[Bacterial
Inoculations](https://www.google.com/url?q=https://drive.google.com/file/d/1MPZyq4kKyrxe01y69Wl0KE2fwnsOoe-F/view?usp%3Dsharing&sa=D&source=editors&ust=1634875622036000&usg=AOvVaw3u12XqcjyHXzqN0wQGFJpC)

For inoculations, the media used was liquid LB broth with
carbenicillin.

[Miniprep](https://www.google.com/url?q=https://drive.google.com/file/d/1-01LD0ba7ivXSFTz1Be_RjfLu_rdywPc/view?usp%3Dsharing&sa=D&source=editors&ust=1634875622037000&usg=AOvVaw050eeKsK-DJPq0scMnUGLq)

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://www.google.com/url?q=https://drive.google.com/open?id%3D1Vi2QSl52If8v-YGc1Duw3NxY4bmQcJ2E&sa=D&source=editors&ust=1634875622038000&usg=AOvVaw1e7ZdCzIUKPoY9QnF_mq9h)

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://www.google.com/url?q=https://drive.google.com/open?id%3D1y5LhMLLkGVLBvMsvIWSjNnUJInYp1hT5&sa=D&source=editors&ust=1634875622039000&usg=AOvVaw088jjo4sJIQAZy5S-VA1U3)

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://www.google.com/url?q=https://drive.google.com/drive/folders/12iHE53T5bKEGZIH8FnU9nrHt3hbCaEiP&sa=D&source=editors&ust=1634875622040000&usg=AOvVaw0gO3DMmmSHshsEBNRClcpE)

For this assay, the Pierce Cypridina Luciferase Glow Assay Kit
(Thermo Fisher,
16170)
was used.

---

# Stem Cell Protocol

## hiPSC culture

Green fluorescent protein (GFP) labeled human induced pluripotent
stem cells
(hiPSC)
was obtained 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.

## The generation of cerebral organoids using the microfluidics system

Mainly, we followed the protocol of the STEMdiff Cerebral Organoid
Kit (cat.
#08570),
with slight modifications to some steps. hiPSC-containing droplets were generated on day 0
using a
microfluidics machine, with a relatively high cell density in each droplet (e.g. 100
cell/droplet). In
particular, the droplets were made from 30% volume cell+media suspension in 70% Matrigel to
provide
structural support for the growth of the cells.The droplets were harvested and seeded into
an Ultra-low
Adherent 6 well plate for Suspension Culture (Stemcell, cat. #38071) in 2-3 mL EB Seeding
Medium. Each
well
should contain around 2000-3000 droplets. Proper amounts of media were added on day 2 and 4.
Upon
progression into the induction and differentiation stages (day 5 and 7, respectively), old
media was
removed
with the usage of a cell strainer and new media was dispensed. The ECM-embedding step on day
7 according
to
the original protocol was omitted as the EBs should develop inside the Matrigel droplet.
Supplement all
media with 0.5-1% Matrigel if EBs start to expand outside the droplet surface and fusion
occurs.
Finally, on
day 10, the organoids were plated in maturation media as they enter the maturing stage; the
use of an
orbital shaker is optional as it facilitates organoid fusion. Perform media change every 3-4
days for
the
organoids until they mature after day 40 and are ready for downstream analysis or drug
assays.

## 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 a 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.

Duke iGEM © 2021

Duke iGEM © 2021

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

# Team:Duke/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

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

### Describe what your results mean

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

### Project Achievements

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

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

### Inspiration

See how other teams presented their results.

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