Difference between revisions of "Part:BBa K3185001"
(→Result) |
(→Result) |
||
Line 47: | Line 47: | ||
<br> | <br> | ||
<br> | <br> | ||
− | [[File:SPYC-T timeD | + | [[File:SPYC-T timeD.png|300px|thumb|left|Fig. 12 Quantification of conjugated band |
<br> | <br> | ||
Conjugated bands’ intensity was quantified with ImageJ. Orange dots show averages value of three experiments. Blacklines show standard deviations. The time point 60min was deleted because it includes negative value. | Conjugated bands’ intensity was quantified with ImageJ. Orange dots show averages value of three experiments. Blacklines show standard deviations. The time point 60min was deleted because it includes negative value. |
Revision as of 10:26, 19 October 2019
Tm Encapsulin
Usage and Biology
TmEncapsulin is a protein found from Thermotoga maritima. A paper says that it consists of 60 monomers and forms capsule, Virus-like particle(VLP)[1]. iGEM also treats it as a useful part (BBa_K192000).
We used TmEncapsulin as biological polymer, because it consists of 60 monomers. Also, this has three tag sites. First is 6×His-tag inserted in the C-terminus of TmEncapsulin for protein purification. Second is HA-tag inserted between TmEncapsulin and 6x-His tag to detect it by using the antibody. Third is a 6x-His tag inserted between the C-terminus of Encapsulin and 6x-His tag because, in a paper, it is said that 6x-His tag inserted in the C-terminus of Encapsulin is not presented on the surface of Encapsulin well, so it can’t bind to Ni-NTA Agarose beads .In the same paper, it is also said that heat-resistance is improved when inserting 6x-His tag and linker between #43 and #44 amino acids of native encapsulin, so we designed like that[2].
We put it between BamHI site and Ndel site on pET11-a. We used BL21 (DE3) for gene expression. We used Ni-NTA Agarose beads for purification. After that, we confirmed molecular weight of TmEncapsulin 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 492 - 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
Result
References
1 Lukarska, M., Fournier, G., Pflug, A., Resa-Infante, P., Reich, S., Naffakh, N., and Cusack, S. (2017).
Structural basis of an essential interaction between influenza polymerase and Pol II CTD.
Nature 541, 117–121.
2 Moon, H., Lee, J., Min, J., and Kang, S. (2014).
Developing genetically engineered encapsulin protein cage nanoparticles as a targeted delivery nanoplatform.
Biomacromolecules 15, 3794–3801.