Difference between revisions of "Part:BBa K1668008"
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We construct the device tcdA1 to tandem express toxic protein tcdA1 and reporter mCherry. Toxic protein tcdA1 is used to kill termites in our project. | We construct the device tcdA1 to tandem express toxic protein tcdA1 and reporter mCherry. Toxic protein tcdA1 is used to kill termites in our project. | ||
| − | tcdA1, one of the biggest proteins in bacteria, is first found in Photorhabdus luminescens. It forms channels and assists other toxins across the cell membrane1. It belongs to tc toxic protein family, which is widely distributed among different gram-negative and gram-positive bacteria. | + | tcdA1, one of the biggest proteins in bacteria, is first found in Photorhabdus luminescens. It forms channels and assists other toxins across the cell membrane1. It belongs to tc toxic protein family, which is widely distributed among different gram-negative and gram-positive bacteria. tcdA1 can keep the ability to form a pore in a large pH range (from 4 to 11). To be noticed, at pH11, the pore-forming activity of tcdA1 is more than 100-fold greater than at pH6. As the midguts of most insects are alkaline, tc toxic proteins are effective by feeding on insects, including termites.In 2013, the structure of tcdA1 was revealed by researchers and reported in nature(1). |
We clone and standardize the gene into standard plasmid pSB1C3. After confirmation of digestion and sequencing, we transform the plasmid into E.coli BL21(DE3) to achieve better expression level. Despite we observe that transformants have obviously turned red, we didn’t figure out the expected protein band in SDS-PAGE. Judging that the protein is considerably huge in bacteria, more improvements are needed. | We clone and standardize the gene into standard plasmid pSB1C3. After confirmation of digestion and sequencing, we transform the plasmid into E.coli BL21(DE3) to achieve better expression level. Despite we observe that transformants have obviously turned red, we didn’t figure out the expected protein band in SDS-PAGE. Judging that the protein is considerably huge in bacteria, more improvements are needed. | ||
Revision as of 12:39, 14 September 2015
tcdA1-device
We construct the device tcdA1 to tandem express toxic protein tcdA1 and reporter mCherry. Toxic protein tcdA1 is used to kill termites in our project.
tcdA1, one of the biggest proteins in bacteria, is first found in Photorhabdus luminescens. It forms channels and assists other toxins across the cell membrane1. It belongs to tc toxic protein family, which is widely distributed among different gram-negative and gram-positive bacteria. tcdA1 can keep the ability to form a pore in a large pH range (from 4 to 11). To be noticed, at pH11, the pore-forming activity of tcdA1 is more than 100-fold greater than at pH6. As the midguts of most insects are alkaline, tc toxic proteins are effective by feeding on insects, including termites.In 2013, the structure of tcdA1 was revealed by researchers and reported in nature(1).
We clone and standardize the gene into standard plasmid pSB1C3. After confirmation of digestion and sequencing, we transform the plasmid into E.coli BL21(DE3) to achieve better expression level. Despite we observe that transformants have obviously turned red, we didn’t figure out the expected protein band in SDS-PAGE. Judging that the protein is considerably huge in bacteria, more improvements are needed.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
Illegal NheI site found at 8257
Illegal NheI site found at 8590 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1651
Illegal BamHI site found at 1144 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 7691
Illegal NgoMIV site found at 8351
Illegal AgeI site found at 979
Illegal AgeI site found at 3527 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961