Difference between revisions of "Part:BBa K371019"
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| − | This composite consists of eight pdu bacterial microcompartment(BMC) structure gene following a natural lac promotor(BBa_R0010). These genes come from four basic parts,pduAB,pduJK,pduN and pduTUV, which consist of the natural RBS and gene separately. | + | This composite consists of eight pdu bacterial microcompartment(BMC) structure gene following a natural lac promotor(BBa_R0010). These genes come from four basic parts,pduAB,pduJK,pduN and pduTUV, which consist of the natural RBS and gene separately. This part alone can assemble an empty BMC shell complex, which can be used as a platform to ''de novo'' make artificial organelles with different functions can be constructed. With enzymes or binding proteins inside the shell, different nanoreactors or nanoreservoirs can be produced. |
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| + | In the expression of protein, we use and unite the method mentioned in the previous papers. [see protocol] The bacterial was induced by 1 mM IPTG at 30 ℃ for 4 hours. The results was as follows.[the one without pduN was a negative control used to prove the function of pduN: in the growth process, the one without pduN tends to grow with more deposit implying its possible role in the vertex] | ||
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| + | [[Image:Before_iptg..jpg]] | ||
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| + | We use the ultracentrifuge to purify the proteins, with 48000 g speed, we get our targeted proteins in the pellet. | ||
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| + | [[Image:Pdu_Sds-page.jpg]] | ||
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| + | We also try to see pdu BMC directly by eyes through the technique of transmission electron microscope(TEM). ''Escherichia coli'' with this parts were fixed in Glutardialdehyde in PBS, stained in in 1% osmium tetroxide. Then after several steps to dehydration and embed by resin, the sample were thin sectioned and post-stained with uranyl acetate and lead citrate to get them ready for observation. Here is the result of electron microscope. | ||
| + | [[Image:2010ustc-dj4.JPG|600px]] | ||
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| + | The pdu BMC was located in ''E.coil'' a special spacial pattern: the BMC all line up, evenly space, down the central axis of the rod-shaped bacteria. | ||
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| + | [[Image:2010-USTC-DJ3.JPG|600px| Electron microscope]] | ||
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| + | This image is a partial enlarged view of the pdu BMC. Unfortunately, we can not see the details of its protein complex structure. | ||
Latest revision as of 08:32, 31 October 2010
pdu(Propanediol utilization)BMC shell gene under Plac control
This composite consists of eight pdu bacterial microcompartment(BMC) structure gene following a natural lac promotor(BBa_R0010). These genes come from four basic parts,pduAB,pduJK,pduN and pduTUV, which consist of the natural RBS and gene separately. This part alone can assemble an empty BMC shell complex, which can be used as a platform to de novo make artificial organelles with different functions can be constructed. With enzymes or binding proteins inside the shell, different nanoreactors or nanoreservoirs can be produced.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1894
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 3236
- 1000COMPATIBLE WITH RFC[1000]
In the expression of protein, we use and unite the method mentioned in the previous papers. [see protocol] The bacterial was induced by 1 mM IPTG at 30 ℃ for 4 hours. The results was as follows.[the one without pduN was a negative control used to prove the function of pduN: in the growth process, the one without pduN tends to grow with more deposit implying its possible role in the vertex]
We use the ultracentrifuge to purify the proteins, with 48000 g speed, we get our targeted proteins in the pellet.
We also try to see pdu BMC directly by eyes through the technique of transmission electron microscope(TEM). Escherichia coli with this parts were fixed in Glutardialdehyde in PBS, stained in in 1% osmium tetroxide. Then after several steps to dehydration and embed by resin, the sample were thin sectioned and post-stained with uranyl acetate and lead citrate to get them ready for observation. Here is the result of electron microscope.
The pdu BMC was located in E.coil a special spacial pattern: the BMC all line up, evenly space, down the central axis of the rod-shaped bacteria.
This image is a partial enlarged view of the pdu BMC. Unfortunately, we can not see the details of its protein complex structure.