Difference between revisions of "Part:BBa K1111012:Experience"
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This part can be used in any experience with biotin. Biotinylation of biological material is widely used and quick to do. Once biotinylated, the molecule will be able to attach to the streptavidin expressed in bacteria. For us, the material was nanoparticles, that we nicely biotinylated ([http://2013.igem.org/Team:EPF_Lausanne/Nanoparticles nanoparticles protocol and usage]) | This part can be used in any experience with biotin. Biotinylation of biological material is widely used and quick to do. Once biotinylated, the molecule will be able to attach to the streptavidin expressed in bacteria. For us, the material was nanoparticles, that we nicely biotinylated ([http://2013.igem.org/Team:EPF_Lausanne/Nanoparticles nanoparticles protocol and usage]) | ||
==Sequencing== | ==Sequencing== | ||
| − | We sequenced this part once the Gibson assembly made. To do so, we used primers for iGEM sites VF2 and VR, in order to sequence all that was inserted in the backbone pSB1C3. | + | We sequenced this part once the Gibson assembly made. To do so, we used primers for iGEM sites VF2 and VR, in order to sequence all that was inserted in the backbone pSB1C3. The sequencing results were aligned to the ''theoretical'' coding sequences using ''ClustalW2'' program [http://www.ebi.ac.uk/Tools/msa/clustalw2/]. |
| − | The sequencing results | + | In the following pdf, the starts mean 100% matching sequences. |
| − | <br>[[ | + | <br>VF2 sequencing results of INP_Streptavidin Alive for two colonies we picked: |
| + | <br>[[Image: Team-EPF-Lausanne_ClustalW2_VF2_col1%262_ISA.pdf ]] | ||
| + | <br>VR sequencing results of INP_Streptavidin Alive for two colonies we picked: | ||
| + | <br>[[Image: Team-EPF-Lausanne_ClustalW2_VR_col1%262_ISA.pdf ]] | ||
| + | <br>Note: you can notice that there is no overlap between the two sequencing results but thanks to another forward primer (5'- <font color= red>AATAATATGGCCGACCATTG </font>-3') we designed, we were able to confirm the sequences. | ||
==Microscopy== | ==Microscopy== | ||
Also, we transformed cells and used them to do microscopy. Two experiments were done, one with a fluorescent antibody against streptavidin and the other one with a fluorescent biotin. We did both in case the antibody would be too big, since biotin is a small molecule best suit to cross glycocalix. | Also, we transformed cells and used them to do microscopy. Two experiments were done, one with a fluorescent antibody against streptavidin and the other one with a fluorescent biotin. We did both in case the antibody would be too big, since biotin is a small molecule best suit to cross glycocalix. | ||
| − | + | ==User Reviews== | |
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Latest revision as of 23:04, 2 October 2013
This experience page is provided so that any user may enter their experience using this part.
Please enter
how you used this part and how it worked out.
Applications of BBa_K1111012
This part can be used in any experience with biotin. Biotinylation of biological material is widely used and quick to do. Once biotinylated, the molecule will be able to attach to the streptavidin expressed in bacteria. For us, the material was nanoparticles, that we nicely biotinylated ([http://2013.igem.org/Team:EPF_Lausanne/Nanoparticles nanoparticles protocol and usage])
Sequencing
We sequenced this part once the Gibson assembly made. To do so, we used primers for iGEM sites VF2 and VR, in order to sequence all that was inserted in the backbone pSB1C3. The sequencing results were aligned to the theoretical coding sequences using ClustalW2 program [http://www.ebi.ac.uk/Tools/msa/clustalw2/].
In the following pdf, the starts mean 100% matching sequences.
VF2 sequencing results of INP_Streptavidin Alive for two colonies we picked:
File:Team-EPF-Lausanne ClustalW2 VF2 col1&2 ISA.pdf
VR sequencing results of INP_Streptavidin Alive for two colonies we picked:
File:Team-EPF-Lausanne ClustalW2 VR col1&2 ISA.pdf
Note: you can notice that there is no overlap between the two sequencing results but thanks to another forward primer (5'- AATAATATGGCCGACCATTG -3') we designed, we were able to confirm the sequences.
Microscopy
Also, we transformed cells and used them to do microscopy. Two experiments were done, one with a fluorescent antibody against streptavidin and the other one with a fluorescent biotin. We did both in case the antibody would be too big, since biotin is a small molecule best suit to cross glycocalix.
User Reviews
UNIQe6030345a72ff339-partinfo-00000000-QINU UNIQe6030345a72ff339-partinfo-00000001-QINU