Team:MIT/results

Results

Graphs, explanation of results

Interpreting the Results of Cytometry

We used CytoFlow, a software developed by our iGEM advisor, Brian Teague, to analyze the results of our HEK transfections. Import samples from file generated by the flow cytometer.

1. Assign each sample a unique set of conditions, which could be Booleans, Categories or Numbers. These distinctions can be taken from your transfection planning document. Name carefully, as these variable will be what you use to process your data later

2. Gate the live cells based off their forward scatter height (FSC-H) and side scatter width (SSC-W). These are the laser scatters caused by the cells passing through the cytometer. You want to select a population of cells that have a high FSC-H and SSC-W. [Example figure goes here]

3. Set your transfection threshold, by measuring the amount of fluorescence outputted by your transfection marker. Transfection efficiencies typically range from approximately 10-85%. [Example figure goes here]

4. Bin your cells by the amount of plasmid in each cell. Binning is a method of standardizing your output across different cell plasmid concentrations. If a cell receives more plasmid, we would expect it to have a higher fluorescence output across all colors. We divided our population into 40 transfection bins. [Example figure goes here]

5. Based on your variable and output fluorescence, you want to group your cells by variables and compare the fluorescence in every case.

ASO and MS2 Results

Before testing the tiling of our ASO guides, we needed to determine the ideal amount of mKate, Ms2, and ASO required to see an effect on our sytem.

mKate Titration

We varied the amounts of mKate-ff4 from 10 to 500 ng using ASO3 and ASO2+. The ASO2+ was co-transfected with Ms2.

[Figure ASO3vsmkateamt_red][Figure ASO2plusvsmkateamt_red]

Caption: mKate-ff4 reporter amounts (10ng-500ng) vs. the amount of red fluorescence (AU). The color of the line indicate the transfection bin of each result.

For the mkate titration for ASO3, we observe a standard mkate curve for all reporter concentration above 100 ng. Below 100 ng there was a disturbance in the normal curve, indicating that our system might be acting on the system.

For the mkate titration for ASO2+, we also observe a disturbance at 100 ng of reporter.

Based on these results, we decided to transfect with 100ng of mkate-ff4 moving forward.

TRE:Ms2 DOX Induction

In order to determine the effects of Ms2 on our system, we put Ms2 downstream of a doxcycline (DOX) promoter. This allowed us to vary the amount of Ms2 in the system based on the amount of DOX added. We ran DOX inductions for ASO0+, ASO2+ and ASO4+.

[Figure ASO0plusdoxms2_red911][Figure ASO4plusdoxms2_red 911]
[Figure ASO2plusdoxms2_red911]

Caption: DOX amounts (5uM-500uM) vs. the amount of red fluorescence (AU). The color of the line indicate the transfection bin of each result.

Results for ASO0+ showed that as levels of DOX, and therefore Ms2 levels, rose in the system, there was a decrease in red fluorescence. Results for ASO2+ show a slight increase in red across DOX concentrations. Results for ASO4+ show a slight decrease in red across DOX concentrations.

The results from ASO0+ show a log decrease in DOX concentrations, indicating that our system is functioning as expected. Based on these results we concluded that maximizing the amount of Ms2 in our system was preferable. Therefore, we decided to transfect with 300 ng of Ms2 moving forward, as this was the maximum amount of DNA per plasmid we transfected.

ASO titration

We titrated the amount of ASO1 and ASO1+ from 10 to 200 ng. ASO1+ was co-transfected with Ms2.

[Figure ASO1amt_red_no Ms2] [Figure ASO1plusamt_red_Ms2]

Caption: ASO amounts (20ng-200ng) vs. the amount of red fluorescence (AU). The color of the line indicate the transfection bin of each result.

In both results there was no decrease across the ASO levels. We concluded that 200 ng was not enough to have an effect on our system. Moving forward, we transfected with 300 ng of ASO.

After optimizing the levels of ASO, Ms2 and mKate-ff4 we tested our tiled guides.

Tiling experiments

We transfected ASOs 0, 1, 2, and 3 by themselves. We transfected ASOs 0+, 1+, 2+, 3+, and 4+ with Ms2. An non-targeting ASO (labeled J) was transfected as a control.

[Figure ASO0to3_junk_red] [Figure ASO0to4plusms2_junk_red]

ASO 0, 1, and 3 had a knockdown in red from the control, with ASO3 showing the greatest decrease. ASO 2 had similar levels of red as the control.

Unexpectedly, the ASO+s had an increase in red from the control. Our hypothesis was that too much mKate was being produced before the ASOs and Ms2 had enough time to be translated. We did further experiments to add a time delay to the production of mKate.

mKate DOX Induction

ASOs and Ms2 need to be produced by the cell before they are able to affect the splicing of mKate-ff4. In order to add a time delay between when they were being produced and when mKate was being produced, we put mKate downstream of a DOX inducible promoter. DOX was added to the system 24 hours after transfection. We used ASO1 and ASO1+, and a well with just mKate for control.

[Figure ASO1_mkateDOX] [Figure ASO1plusMs2_mkateDOX][Figure mkate_induction]

Unfortunately, there was no significant difference between the control titration and the ASO or ASO+ titration. All the ASOs will have to be tested in this way before determining whether or not this system is effective.

Guide and Cas13a Results

Before testing the tiling of our Cas13a guides, we needed to determine the ideal amount of mKate and guide required to see an effect on our sytem.

mKate Titration

We varied the amounts of mKate-ff4 from 10 to 500 ng using Guide2. Cas13a was used.

[Figure cas13_g2_mkatetitration2] [Figure cas13_noguide_mkatetitration2][Figure mkate_titration]

Caption: mKate-ff4 reporter amounts (10ng-500ng) vs. the amount of red fluorescence (AU). The color of the line indicate the transfection bin of each result.

We observe a standard mkate curve for all reporter concentration above 100 ng. At 100 ng there was a disturbance in the normal curve, indicating that our system might be acting on the system.

Based on these results, we decided to transfect with 100ng of mkate-ff4 moving forward.

Guide Titration

We titrated the amount of Guide2 from 10 to 500 ng. Guide2 was co-transfected with cas13a and dcas13a.

[Figure cas13_g2_guidetitration] [Figure dcas13_g2_guidetitration]

Caption: Guide amounts (10ng-500ng) vs. the amount of red fluorescence (AU). The color of the line indicate the transfection bin of each result.

In both results there was a decrease in mKate when 10ng of guide was added to the system. Moving forward, we transfected with 10 ng of guide.

After optimizing the levels of guide and mKate-ff4 we tested our tiled guides:

Guide Tiling

We transfected Guide 1, 2, 3, 4 and 5 with dCas13a and dCas13a-ddx6. A non-targeting junk guide was used for control (labeled 0 on the graph)

[Figure Guide_tiling_dcas] [Figure Guide_tiling_dcas-ddx6]

In the dCas13-ddx6 experiment there was no knockdown of mKate across the guides. In the dCas13a, there was minimal knockdown. The greatest knockdown occurred in the presence of Guide2. Our hypothesis about the reason we were not seeing the knockdown we expected was that too much mKate was being produced before the guides and dCas13a had enough time to be translated. We did further experiments to add a time delay to the production of mKate.

mKate DOX Induction

Guides and dCas13a needs to be produced by the cell before they are able to affect the splicing of mKate-ff4. In order to add a time delay between when they were being produced and when mKate was being produced, we put mKate downstream of a DOX inducible promoter. DOX was added to the system 24 hours after transfection. We used Guide3, and a non-targeting junk guide as a control.

[Figure Guide3_mkate_Dox] [Figure JunkGuide_mkate_dox] [Figure mKate_induction1010]

Unfortunately, there was no significant difference between the control titration and the Guide3 or Junk guide induction. However, even the control in this experiment was not showing clear results, so the experiment should be rerun before drawing conclusions about the system. All the guides will have to be tested in this way before determining whether or not this system is effective.

2-exon mKate-HBG reporter

mKate titration

We intended to test our mKate-HBG reporter in the same way as our mKate-ff4 reporter, by increasing the amoung of mKate-HBG from 10-500ng. However upon analysing the results of the reporter independently, we saw that it did not produce fluorescence above baseline.

[Figure mkateHBG_noRBP]

Unfortunately, we were unable to test any of our constructs against this reporter, because the reporter was no producing red.