Difference between revisions of "Team:Gifu/Experiment-page"
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<a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_K1859026" > [BBa_K1859026] </a> | <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_K1859026" > [BBa_K1859026] </a> | ||
− | ", inside | + | ", inside Ⅰ |
<a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_K1859024" > [BBa_K1859024] </a> | <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_K1859024" > [BBa_K1859024] </a> | ||
− | , and inside | + | , and inside Ⅱ |
<a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_K1859025" > [BBa_K1859025] </a> | <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_K1859025" > [BBa_K1859025] </a> | ||
to <i>E. coli</i>, and pre-incubated each in 37℃ over night. Then we cultured with shaking the pre-culture liquids 100 µL with LB-Cm 10 mL in 37 for four hours and extracted total RNA from them with RNA extraction kit. | to <i>E. coli</i>, and pre-incubated each in 37℃ over night. Then we cultured with shaking the pre-culture liquids 100 µL with LB-Cm 10 mL in 37 for four hours and extracted total RNA from them with RNA extraction kit. | ||
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Amino acid chains synthesized from circular mRNA are constructed from repetition in amino acid of target protein, amino acid from ribozyme and amino acid from RBS. In this experiment, we inserted linker sequences into inside of both splicing sites. Therefore, protein chains synthesized from circular messenger RNA that include linker sequences repeat linker sequence, amino acid from RBS, target protein, linker sequence and amino acid from ribozyme.<br><br><br> | Amino acid chains synthesized from circular mRNA are constructed from repetition in amino acid of target protein, amino acid from ribozyme and amino acid from RBS. In this experiment, we inserted linker sequences into inside of both splicing sites. Therefore, protein chains synthesized from circular messenger RNA that include linker sequences repeat linker sequence, amino acid from RBS, target protein, linker sequence and amino acid from ribozyme.<br><br><br> | ||
Generally speaking, length of linker is required of one-forth of diameter of target protein. In the case of RFP, that condition is surely cleared by six amino acid, but it is unknown which linkers are appropriate in order to synthesize poly RFP protein. Therefore, we examined some types of amino acids and length of linker and choice the best linker in this experiment.<br> | Generally speaking, length of linker is required of one-forth of diameter of target protein. In the case of RFP, that condition is surely cleared by six amino acid, but it is unknown which linkers are appropriate in order to synthesize poly RFP protein. Therefore, we examined some types of amino acids and length of linker and choice the best linker in this experiment.<br> | ||
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Latest revision as of 20:02, 18 September 2015
linker + | parts name | |
---|---|---|
GGSGGS | 3'side of the intron BBa_K1332005 |
BBa_K1859001 |
GSGSGS | BBa_K1859002 | |
(GGSGGS)×2 | BBa_K1859007 | |
(GSGSGS)×2 | BBa_K1859008 | |
(GGSGGS)×3 | BBa_K1859009 | |
(GSGSGS)×3 | BBa_K1859010 | |
HHHHHH | BBa_K1859003 |
+ linker | parts name | |
---|---|---|
5'side of the intron BBa_K1332003 | GGSGGS | BBa_K1859004 |
GSGSGS | BBa_K1859005 | |
(GGSGGS)×2 | BBa_K1859011 | |
(GSGSGS)×2 | BBa_K1859012 | |
(GGSGGS)×3 | BBa_K1859013 | |
(GSGSGS)×3 | BBa_K1859014 | |
HHHHHH | BBa_K1859006 |
We made following generators by using these parts.
Fig17. Localization of linker sequence after self-splicing
Amino acid chains synthesized from circular mRNA are constructed from repetition in amino acid of target protein, amino acid from ribozyme and amino acid from RBS. In this experiment, we inserted linker sequences into inside of both splicing sites. Therefore, protein chains synthesized from circular messenger RNA that include linker sequences repeat linker sequence, amino acid from RBS, target protein, linker sequence and amino acid from ribozyme.
Generally speaking, length of linker is required of one-forth of diameter of target protein. In the case of RFP, that condition is surely cleared by six amino acid, but it is unknown which linkers are appropriate in order to synthesize poly RFP protein. Therefore, we examined some types of amino acids and length of linker and choice the best linker in this experiment.
We inserted these plasmid into an E. coli and made it synthesize proteins and performed SDS-PAGE by using these proteins. We did SDS-PAGE without boiling to check fluorescence of RFP. Because RFP’s structure is strong, we can see the fluorescence.