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Team:LMU-Munich/Data/gfp spore
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| - | <p align="justify">For creating fusion proteins for the '''Sporo'''beads, the genes ''gfp'', ''cotZ'' and ''cgeA'' were brought into Freiburg Standard | + | <p align="justify">For creating fusion proteins for the '''Sporo'''beads, the genes ''gfp'', ''cotZ'' and ''cgeA'' were amplified and brought into Freiburg Standard. The restriction site NgoMIV was inserted just after the startcodon of the genes of the crust proteins. Since this restriction site adds six additional basepairs the resulting gene is two codons longer ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K823032 CotZ], CgeA). It is not known if this insertion has any effect on protein expression. Therefore we created an additional version in which we deleted the following six basepairs, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823031 CotZ-2aa] and CgeA-2aa. |
| + | <br>Afterwards we first fused both versions of ''cotZ'' to its two native promoters, P<sub>''cotV''</sub> and P<sub>''cotYZ''</sub>, and to P<sub>''cgeA''</sub>, which regulates the transcription of ''cgeA''. For the two ''cgeA'' variants we only used the native promoter P<sub>''cgeA''</sub> and the stonger one of the two promoters of the ''cotVWXYZ'' cluster, P<sub>''cotYZ''</sub> (for more details see [https://2012.igem.org/Team:LMU-Munich/Data/crustpromoters crust promotor evaluation]. While [http://partsregistry.org/Part:BBa_K823039 ''gfp''] was ligated to the terminator B0014 (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0014 Registry]). When these constructs were finished and confirmed by sequencing, we fused them together applying the [http://partsregistry.org/Help:Assembly_standard_25 Freiburg standard] to create in frame fusion proteins, flanked by one of the three promoters and the terminator.This way we created C-terminal fusion proteins. | ||
| + | <br>As we are working with B. subtilis spores, we needed to clone our final constructs into an empty Bacillus vector, so that they could get integrated into the genome of ''B. subtilis'' after transformation. Thus we picked the empty vector pSB<sub>BS</sub>1C from our '''''Bacillus''B'''io'''B'''rick'''B'''ox, for the ''cotZ'' constructs. | ||
<br>All the '''Sporo'''beads were investigated by fluorescence microscopy and analysed with ImageJ and the statistical software '''R'''. The intensity bar charts show the fluorescence intensity, while the 3D graphs illustrate the fluorescence intensity spread across the spore surface, which correlates with the distribution of our fusion proteins. For analysis we measured the fluorescence intensity of a area of 750px per spore by using ImageJ and evaluated it with the statistical software R. The following graph shows the results of microscopy and ImageJ analysis. | <br>All the '''Sporo'''beads were investigated by fluorescence microscopy and analysed with ImageJ and the statistical software '''R'''. The intensity bar charts show the fluorescence intensity, while the 3D graphs illustrate the fluorescence intensity spread across the spore surface, which correlates with the distribution of our fusion proteins. For analysis we measured the fluorescence intensity of a area of 750px per spore by using ImageJ and evaluated it with the statistical software R. The following graph shows the results of microscopy and ImageJ analysis. | ||
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[[File:LMU gfp spore data.png|620px]] | [[File:LMU gfp spore data.png|620px]] | ||
| - | <p align="justify">It appears the strain B70 with the construct [http://partsregistry.org/Part:BBa_K823049 P<sub>''cotYZ''</sub>-''cotZ''<sub>-2aa</sub>-''gfp''-''terminator''] and the deleted native ''cotZ'' had the strongest fluorescence. Thus this would be the strain for future '''Sporo'''beads with special functions. In some 3D graphs the picked cell does not respresent the average fluorescence intensity depicted in the bar chart, because it was chosen randomly.</p> | + | <p align="justify">It appears the strain B70 with the construct [http://partsregistry.org/Part:BBa_K823049 P<sub>''cotYZ''</sub>-''cotZ''<sub>-2aa</sub>-''gfp''-''terminator''] and the deleted native ''cotZ'' had the strongest fluorescence. Thus this would be the strain for future '''Sporo'''beads with special functions. In some 3D graphs the picked cell does not respresent the average fluorescence intensity depicted in the bar chart, because it was chosen randomly. Furthermore it is noitcable that the strain B 45 containing the same construct but without the deletion of the native gene has a strong fluorescence output as well even though it is not as high as the deletion muant. Additionally there is a difference in fluorescence activity between the two versions of ''cotZ'' recognizable. ''CotZ''-2aa is glowing stronger than the other variant in both the cotZ deletion mutant and the wildtype strain </sub></p> |
Revision as of 20:03, 26 September 2012
The LMU-Munich team is exuberantly happy about the great success at the World Championship Jamboree in Boston. Our project Beadzillus finished 4th and won the prize for the "Best Wiki" (with Slovenia) and "Best New Application Project".
[ more news ]
GFP-Sporobead Evaluation
For creating fusion proteins for the Sporobeads, the genes gfp, cotZ and cgeA were amplified and brought into Freiburg Standard. The restriction site NgoMIV was inserted just after the startcodon of the genes of the crust proteins. Since this restriction site adds six additional basepairs the resulting gene is two codons longer (CotZ, CgeA). It is not known if this insertion has any effect on protein expression. Therefore we created an additional version in which we deleted the following six basepairs, CotZ-2aa and CgeA-2aa.
Afterwards we first fused both versions of cotZ to its two native promoters, PcotV and PcotYZ, and to PcgeA, which regulates the transcription of cgeA. For the two cgeA variants we only used the native promoter PcgeA and the stonger one of the two promoters of the cotVWXYZ cluster, PcotYZ (for more details see crust promotor evaluation. While gfp was ligated to the terminator B0014 (see Registry). When these constructs were finished and confirmed by sequencing, we fused them together applying the Freiburg standard to create in frame fusion proteins, flanked by one of the three promoters and the terminator.This way we created C-terminal fusion proteins.
As we are working with B. subtilis spores, we needed to clone our final constructs into an empty Bacillus vector, so that they could get integrated into the genome of B. subtilis after transformation. Thus we picked the empty vector pSBBS1C from our BacillusBioBrickBox, for the cotZ constructs.
All the Sporobeads were investigated by fluorescence microscopy and analysed with ImageJ and the statistical software R. The intensity bar charts show the fluorescence intensity, while the 3D graphs illustrate the fluorescence intensity spread across the spore surface, which correlates with the distribution of our fusion proteins. For analysis we measured the fluorescence intensity of a area of 750px per spore by using ImageJ and evaluated it with the statistical software R. The following graph shows the results of microscopy and ImageJ analysis.
It appears the strain B70 with the construct PcotYZ-cotZ-2aa-gfp-terminator and the deleted native cotZ had the strongest fluorescence. Thus this would be the strain for future Sporobeads with special functions. In some 3D graphs the picked cell does not respresent the average fluorescence intensity depicted in the bar chart, because it was chosen randomly. Furthermore it is noitcable that the strain B 45 containing the same construct but without the deletion of the native gene has a strong fluorescence output as well even though it is not as high as the deletion muant. Additionally there is a difference in fluorescence activity between the two versions of cotZ recognizable. CotZ-2aa is glowing stronger than the other variant in both the cotZ deletion mutant and the wildtype strain </sub>
