iGEM 2018 — Team McGill

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# Team:McGill/Notebook

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# Notebook

###### Week of June 18-24 2018

* Miniprep + digest to confirm GCIR1 ligation

###### Week of August 6-12 2018

* Double digest on DNA + gel electrophoresis

###### Week of August 13-19 2018

* Verify synnotch ligation via gel electrophoresis
* Maxiprep of the synnotch plasmid from successful colonies
* Tissue Culture: thawed and passaged HEK293 cells + NMuMG cells

###### Week of August 27-September 2 2018

* Cell lysis of transformed HEK293 cells
* Bradford assay on cell sample lysates
* Western blot

###### Week of September 17-23 2018

* Exposed NMuMG synnotch western blot
* Seeded HEK293 cells for transfection + froze down parental cells not used in flow cytometry
* Transfected HEK293 cells + NMuMG cells
* Performed fluorescence activated cell sorting (FACS) on transfected cells


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# Team:McGill/Experiments

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# Experiments and Protocols

##### Miniprep of Plasmid DNA (Adapted from the protocol from the Qiagen plasmid mini kit : cat. nos. 12123))

* Harvest overnight bacterial culture by centrifuging at 6000 x g for 15 min at 4°C.
* Resuspend the bacterial pellet in 0.3 mL buffer P1 (from the Qiagen plasmid mini-prep kit cat. nos. 12123)
* Add 0.3 mL of buffer P2 and mix thoroughly by vigorously inverting 4–6 times. Incubate at room temperature for 5 min.
* Add 0.3 mL of pre chilled buffer P3 and mix thoroughly by vigorously inverting 4–6 times. Incubate on ice for 5 minutes.
* Centrifuge at 16000xg for 10 minutes at 4 °C. Recentrifuge if the supernatant is not clear.
* Equilibrate a 20 QIAGEN-tip by applying 1 mL of buffer QBT, and allow the column to empty by gravity flow.
* Apply the supernatant from the centrifugation step to the QIAGEN-tip and allow it to enter the resin by gravity flow.
* Wash the QIAGEN-tip with twice with 2 mL of buffer QC. Allow Buffer QC to move through the QIAGEN-tip by gravity flow.
* Elute the DNA with 0.8 mL of buffer QF into a clean 2 mL vessel.
* Precipitate DNA by adding 0.56 ml of isopropanol to the eluted DNA. Centrifuge at 16000xg for 30 minutes at 4°C.
* Wash the DNA pellet with 1 mL of room temperature 70% ethanol and centrifuge at 16000xg for 10 minutes. Decant the supernatant.
* Air-dry the pellet for 10 minutes and redissolve the DNA at a suitable concentration in TE buffer.

##### Maxiprep of Plasmid DNA (Adapted from the protocol from the Qiagen plasmid maxi kit (cat. nos. 12162))

* Harvest overnight bacterial culture by centrifuging at 6000 x g for 15 min at 4°C.
* Resuspend the bacterial pellet in 10 mL buffer P1 (from the Qiagen plasmid mini-prep kit cat. nos. 12123)
* Add 10 mL of buffer P2 and mix thoroughly by vigorously inverting 4–6 times. Incubate at room temperature for 5 min.
* Add 10 mL of pre chilled buffer P3 and mix thoroughly by vigorously inverting 4–6 times. Incubate on ice for 20 minutes.
* Centrifuge at 20000xg for 30 minutes at 4 °C. Recentrifuge if the supernatant is not clear.
* Equilibrate a 500 QIAGEN-tip by applying 10 mL of buffer QBT, and allow the column to empty by gravity flow.
* Apply the supernatant from the centrifugation step to the QIAGEN-tip and allow it to enter the resin by gravity flow.
* Wash the QIAGEN-tip with twice with 30 mL of buffer QC. Allow Buffer QC to move through the QIAGEN-tip by gravity flow.
* Elute the DNA with 15 mL of buffer QF into a clean 2 mL vessel.
* Precipitate DNA by adding 10.5 ml of isopropanol to the eluted DNA. Centrifuge at 16000xg for 30 minutes at 4°C.
* Wash the DNA pellet with 5 mL of room temperature 70% ethanol and centrifuge at 16000xg for 10 minutes. Decant the supernatant.
* Air-dry the pellet for 10 minutes and redissolve the DNA at a suitable concentration in TE buffer.

##### Transfection of Adherent Cells using Effectene reagent (Qiagen)

* The day prior to transfection, seed 2–8 x 10^5 cells per 60 mm dish in 5 ml of growth medium containing FBS and antibiotics.
* Incubate the cells in normal growth conditions. The dishes should be around 60% confluent on the day of transfection.
* On the day of transfection, dilute 1 μg DNA dissolved in TE buffer, pH 7 to pH 8 (minimum DNA concentration: 0.1 μg/μl) with the DNA-condensation buffer, Buffer EC, to a total volume of 150 μl. Add 8 μl Enhancer and mix by vortexing for 1 s.
* Incubate at room temperature for 2–5 min then spin down the mixture for a few seconds to remove drops from the top of the tube.
* Add 25 μl of Effectene Transfection Reagent to the DNA-Enhancer mixture. Mix by pipetting up and down 5 times, or by vortexing for 10 s.
* Incubate the samples for 10 min at room temperature to allow the formation of the transfection-complex.
* While complex formation takes place, aspirate the growth medium from the plate, and wash cells once with 4 ml PBS. Add 4 ml of fresh growth medium containing FBS and antibiotics to the cells.
* Add 1 ml growth medium to the tube containing the transfection complexes. Mix by pipetting up and down twice, and immediately add the transfection complexes drop-wise onto the cells in the 60 mm dishes. Gently swirl the dish to ensure uniform distribution of the transfection complexes.
* Incubate the cells with the transfection complexes under their normal growth conditions for an appropriate time for expression of the transfected gene. .

##### Western Blot

* Cells were first lysed:
  + The media around the cells was aspirated. The cells were then washed twice with chilled PBS and the culture flask was placed on ice.
  + Chilled lysis buffer (150 mM sodium chloride, 1.0% Triton X-100, 50 mM Tris pH 8.0) was then added (0.5 mL per T75 flask). The flask was left on ice for another 10 minutes.
  + A plastic cell scraper was then used to scrape the adherent cells off the flask. The cell suspension was then transferred to centrifuge tubes.
  + The cell suspension was centrifuged for 10 minutes at 4°C at 12000 rpm.
  + The supernatant was decanted and the lysate was transferred to clean centrifuge tubes.* A bradford assay was then performed to determine how much protein to load.
  * An SDS-PAGE gel was prepared to which equal amounts of protein were then loaded in along with a molecular weight marker. In total, around 25 μg of protein from cell lysate was added.
  * The gel was then run at 100 V for 1-2 hours.
  * The gel was transferred to the nitrocellulose membrane:
  + A nitrocellulose membrane was blocked overnight at 4°C using blocking buffer.
  + The membrane was then incubated with appropriate dilutions of primary antibody solution in blocking buffer overnight at 4°C.
  + The membrane was washed thrice with TBST for 5 minutes each.
  + Excess reagent was removed and the membrane was covered in transparent plastic wrap.* The membrane was then analyzed for the protein of interest.

##### Bradford Assay

* A standard curve was prepared by measuring the absorbance at 595 nm of different known protein concentrations.
* Cell lysate were diluted such that they have absorbance values on the standard curve (usually a 1:10 dilution)
* The different cell lysates were added to different wells on a 96 well plate (with duplicates)
* Bradford reagent was then added to the wells (at 5x conc leading to around 200 uL/well
* The 96 well plate was then incubated for 5 minutes at room temperature.
* The plate absorbance was read at 595 nm.

##### Flow Cytometry (FACS)

* Cells were harvested, washed, and suspended at a concentration of 5 x 10^6 cells/mL in a chilled solution of PBS containing 10% FCS and 1% sodium azide. The cells were then transferred to polystyrene round bottom 12 x 75 mm^2 Falcon tubes.
* 10 ug/mL of conjugated primary antibody was then added to the cells. The cells were incubated for 30 minutes in the dark at room temperature.
* The cells were then washed thrice by centrifugation at 400xg for 5 minutes and resuspended in 1 mL of PBS containing 10% FCS and 1% sodium azide. The cells were kept in the dark on ice prior to analysis.
* The cells were then analyzed on a flow cut-meter.

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# Team:McGill/Results

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# Our Results

### Synnotch Expression Confirmation by Fluorescence-activated Cell Sorting (FACS)

![](https://static.igem.org/mediawiki/2018/thumb/1/1a/T--McGill--synnnotchFACS.png/800px-T--McGill--synnnotchFACS.png)

Figure 1. To prepare the cells for FACs, activation of the circuit was induced by the addition of anti-myc antibody and induction of cellular stress mechanically via repeated up and down pipetting. The cells were then incubated for 12 hours and prepared for analysis. Primary mouse anti-myc antibody and secondary Alexa Fluor 488 anti-mouse was used to detect synnotch expression on our target population of cells. Control cells were untransformed HEK 293 cells. Unstained and secondary only controls were used for each population as a further control. As we can see in figure 1, the five controls all showed similar geometric means while the transformed HEK 293 cells with primary and secondary showed a geometric mean approximately 3-fold higher, indicating successful expression of SynNotch on the surface of our target population.

### Synnotch Expression Confirmation by Western Blot

![](https://static.igem.org/mediawiki/2018/5/55/T--McGill--westernblot_figure.png)

Figure 2. Confirmation by Western Blot of Synnotch Expression. The negative control is EV (empty Vector) and shows no bands around the 75-100kDa region where our target protein is predicted to be. The positive control is a Myc-expressing cell line, to show that the anti-Myc antibody is functioning properly. Indeed strong banding shows that the antibody works and is specific. Our synnotch-expressing cells show a pattern similar to the EV negative control but with an additional band directly below 100kDa, which is compatible with the predicted molecular weight of SynNotch (94kDa).

### Basal Expression of GFP by GCIR Transformed Cells

![](https://static.igem.org/mediawiki/2018/thumb/0/0a/T--McGill--GCIRresults.png/800px-T--McGill--GCIRresults.png)

Figure 3. Basal expression of GFP by GCIR1 transformed HEK 293 cells. GFP was detected in its natively fluorescing channel via flow cytometry. HEK 293 cells transformed with empty vectors and transformed with Synnotch and an empty GCIR1 vector showed low baseline GFP. However, both the unstimulated SynNotch GCIR1 double transformants and GCIR1 transformants with empty SynNotch vector show geometric means 8-fold higher than the controls and similar to that of the myc stimulated SynNotch GCIR1 double transformants. There are no significant differences between the fluorescence signitures of these three populations, indicating that the GCIR1 promoter is constitutively active.

### Appendix/Supporting Figures

![](https://static.igem.org/mediawiki/2018/thumb/1/1c/T--McGill--results2.png/799px-T--McGill--results2.png)

Figure 3. Gating strategy for flow cytometry data. The samples were first gated in order to cut off debris that is smaller than expected cell size. By forming this gating strategy, we were able to select for single cells in our FACS experiments.