Team:Newcastle University/Conclusions

From 2008.igem.org

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To analyse the results of the wet lab transformations of the inserts into ''B. subtilis'', we used two methods: [[fluorescence microscopy]] and flow cytometry.
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To analyse the results of the wet lab transformations of the inserts into ''B. subtilis'', we used two methods: microscopy and flow cytometry.
===Microscopy===
===Microscopy===
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===Flow cytometry===
===Flow cytometry===
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Flow cytometry allows us to quantify our results and present them in graphical form.  A sample of cells our engineered Bacillus subtilis cells were injected into the machine which hydro-dynamically focusses the fluid. Lasers are directed onto the stream of fluid, and each particle which passes through the light beam will cause the laser to scatter in a particular way. Fluorescent chemicals are excited to a higher energy state.  
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Flow cytometry allows us to quantify our results and present them in graphical form.  A sample of cells our engineered Bacillus subtilis cells were injected into the machine which hydro-dynamically focusses the fluid. Lasers of a particular wavelength are directed onto the stream of fluid, and each particle which passes through the light beam will cause the laser to scatter in a different way. Cells which absorb and then re-emit the light, result in fluorescence (a higher energy state).
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The detectors in the machine measure the scattering of light and any flourescence which occurs.
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The detectors in the machine measure the scattering of light and any flourescence which occurs, and the data is displayed as histograms and dot plots.
'''0% induction by subtilin''' (i.e in the absence of subtilin): mean flourescence = 7.70
'''0% induction by subtilin''' (i.e in the absence of subtilin): mean flourescence = 7.70
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These results show that the higher the concentraion of subtilin, the more GFP is expressed.
These results show that the higher the concentraion of subtilin, the more GFP is expressed.
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At 10% induction, the subtilin began to cause some of the cells to lyse- this can be seen by the two distinct populations of cells- one being the cells which have lysed, and the other being the remainder of intact cells.  For us, this was a good indication that the subtilin we had collected that day was good quality.
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Revision as of 14:13, 19 September 2008

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Newcastle University

GOLD MEDAL WINNER 2008

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Home >> Results

Results

Microscopy results of iGEMgfp upon 1% induction by subtilin
Microscopy results of iGEMcherry upon 1% induction by subtilin
Flow cytometry results for 0% induction by subtilin.
Flow cytometry results for 1% induction by subtilin.
Flow cytometry results for 10% induction by subtilin.

To analyse the results of the wet lab transformations of the inserts into B. subtilis, we used two methods: microscopy and flow cytometry.

Microscopy

Microscopy work from 08.09.08 showed a difference in the level of flourescence of the iGEMgfp fluorescent cells (higher in 10% subtilin-induced cells compared to 0% subtilin-induced cells). However, there was little difference in the number of cells that fluoresced between the two cultures.

There was no difference in the number of fluorecent cells or the level of flourescence between the 10% subtilin-induced and the 0% subtilin-induced iGEMcherry cells.


Flow cytometry

Flow cytometry allows us to quantify our results and present them in graphical form. A sample of cells our engineered Bacillus subtilis cells were injected into the machine which hydro-dynamically focusses the fluid. Lasers of a particular wavelength are directed onto the stream of fluid, and each particle which passes through the light beam will cause the laser to scatter in a different way. Cells which absorb and then re-emit the light, result in fluorescence (a higher energy state).

The detectors in the machine measure the scattering of light and any flourescence which occurs, and the data is displayed as histograms and dot plots.

0% induction by subtilin (i.e in the absence of subtilin): mean flourescence = 7.70

1% induction by subtilin: mean flourescence = 14.77

10% induction by subtilin: the mean flourescence = 21.95

These results show that the higher the concentraion of subtilin, the more GFP is expressed.

At 10% induction, the subtilin began to cause some of the cells to lyse- this can be seen by the two distinct populations of cells- one being the cells which have lysed, and the other being the remainder of intact cells. For us, this was a good indication that the subtilin we had collected that day was good quality.