EXPERIMENTS

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:

Figure 1. PCR/Cloning Workflow

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

Gel Electrophoresis

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

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

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

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

Bacterial Work

Bacterial Transformation
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

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

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

Western Blot

iBlind Flex Western Protocol

Power Blotter Protocol

MilliporeSigma Protocol

Flow Cytometry

Collecting Cells

Data Analysis

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.