Team:Newcastle University/Conclusions

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[[Image:Montage.jpg|thumb|right|300px|Microscopy results of iGEMgfp upon 1% induction by subtilin]]
[[Image:Montage.jpg|thumb|right|300px|Microscopy results of iGEMgfp upon 1% induction by subtilin]]
[[Image:Montage_pc.jpg|thumb|right|300px|Microscopy results of iGEMcherry upon 1% induction by subtilin]]
[[Image:Montage_pc.jpg|thumb|right|300px|Microscopy results of iGEMcherry upon 1% induction by subtilin]]
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[[Image:Flow1%.jpg|thumb|right|300px|Flow cytometry results for 1% induction by subtilin.]]
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[[Image:Flow10%.jpg|thumb|right|300px|Flow cytometry results for 10% induction by subtilin.]]
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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]].
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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.
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Microscopy work done on 08.09.08 showed a possible difference in the brightness of the iGEMgfp fluorescent cells (brighter in 10% subtilin-induced cells than in 0% subtilin-induced cells). However, there was little difference in the numbers of cells that fluoresced between the two cultures. There was no difference in either the number of fluorecent cells or their brightness between the 10% subtilin-induced and the 0% subtilin-induced iGEMcherry cells.
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===Flow cytometry===
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Microscopy work done on 08.09.08 showed a possible difference in the brightness of the iGEMgfp fluorescent cells (brighter in 10% subtilin-induced cells than in 0% subtilin-induced cells). However, there was little difference in the numbers of cells that fluoresced between the two cultures.
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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 will be excited and emit light at a higher wavelength than the light source.  
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There was no significant difference in either the number of fluorecent cells or their brightness between the 10% subtilin-induced and the 0% subtilin-induced iGEMcherry cells. Both cultures fluoresced very poorly. It was suggested that this could be due to the ''mCherry'' being inherently weak in its fluorecence.
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The detectors in the machine measure the scattering of light and any flourescence which occurs.
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Flow cytometry was performed on the iGEMgfp culture to determine whether the microscopy work did actually show a reporter gene induction by subtilin. This revealed a mean fluorescence of 7.70 counts for 0% subtilin induction, 14.77 counts for 1% subtilin induction and 21.95 counts for 10% subtilin induction. The dramatic increase between each of the measurements suggests that the ncl08 2 component spaRK system + ''gfp'' has inserted correctly into the ''B. subtilis'' chromosomsal DNA and is inducible by subtilin. The fact that two distinct cell populations can be observed in the 10% subtilin induced culture shows that the supernatent added to the cells does indeed contain functional subtilin - one of these populations are the cells that are lysing as a result of the subtilin antibiotic.
 
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Revision as of 13:12, 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 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 work done on 08.09.08 showed a possible difference in the brightness of the iGEMgfp fluorescent cells (brighter in 10% subtilin-induced cells than in 0% subtilin-induced cells). However, there was little difference in the numbers of cells that fluoresced between the two cultures. There was no difference in either the number of fluorecent cells or their brightness 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 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 will be excited and emit light at a higher wavelength than the light source.

The detectors in the machine measure the scattering of light and any flourescence which occurs.


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