Difference between revisions of "Part:BBa K3185000"
0012massan (Talk | contribs) |
0012massan (Talk | contribs) |
||
Line 10: | Line 10: | ||
<br> | <br> | ||
We put it between the BamHI site and the Ndel site on pET11-a. We used BL21 (DE3) for gene expression. We used the Ni-NTA Agarose for purification. After that, we confirmed the molecular weight of SpyCatcher inserted TmEncapusulin by using SDS-PAGE. | We put it between the BamHI site and the Ndel site on pET11-a. We used BL21 (DE3) for gene expression. We used the Ni-NTA Agarose for purification. After that, we confirmed the molecular weight of SpyCatcher inserted TmEncapusulin by using SDS-PAGE. | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
<br> | <br> | ||
<br> | <br> | ||
Line 27: | Line 17: | ||
<!-- --> | <!-- --> | ||
− | = | + | <span class='h3bb'>Sequence and Features</span> |
<partinfo>BBa_K3185000 SequenceAndFeatures</partinfo> | <partinfo>BBa_K3185000 SequenceAndFeatures</partinfo> | ||
− | + | ==Purification== | |
+ | <br> | ||
+ | <h3><font size="4.5">Expression</font> </h3> | ||
+ | <ul> | ||
+ | <li>Cells were grown in 200ml LB media (100μg/ml Ampicillin) at 37<sup>o</sup>C shaking at 140 rpm to an OD600 of 0.5, verifying via a spectrophotometer. | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li>Protein was expressed in 0.1mM IPTG for 2hours. | ||
+ | </ul> | ||
+ | <h3><font size="4.5">SDS-PAGE</font></h3> | ||
+ | <br> | ||
+ | <br> | ||
<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display |
Revision as of 13:50, 17 October 2019
SPYtag inserted Tm Encapsulin
Usage and Biology
TmEncapsulin is a protein found from Thermotoga maritima. A paper (Structural basis of enzyme encapsulation into a bacterial nano compartment) says that it consists of 60 monomers and forms capsule, Virus-like particle(VLP). iGEM also treats it as a useful part (BBa_K192000).
We used TmEncapsulin as a biological polymer. We put Spytag inside TmEncapsulin because we used SpyTag/SpyCatcher system to bind it to other parts. Also, this has three tag and cleavage sites. First is 6×His-tag inserted in the N-terminus of TmEncapsulin for protein purification. Second is HA-tag inserted between TmEncapsulin and 6x-His-tag to detect it by using antibodies. Third is a 6x-His tag because, in the paper, it was used for improving the heat-resistant ability of TmEncapsulin. (Programmable polyproteins built using twin peptide superglues)
We put it between the BamHI site and the Ndel site on pET11-a. We used BL21 (DE3) for gene expression. We used the Ni-NTA Agarose for purification. After that, we confirmed the molecular weight of SpyCatcher inserted TmEncapusulin by using SDS-PAGE.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 77
Illegal BglII site found at 597 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 426
Illegal SapI.rc site found at 457
Purification
Expression
- Cells were grown in 200ml LB media (100μg/ml Ampicillin) at 37oC shaking at 140 rpm to an OD600 of 0.5, verifying via a spectrophotometer.
- Protein was expressed in 0.1mM IPTG for 2hours.
SDS-PAGE