Difference between revisions of "Part:BBa K3185003"
0012massan (Talk | contribs) |
0012massan (Talk | contribs) |
||
| Line 13: | Line 13: | ||
<br> | <br> | ||
<br> | <br> | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
<!-- --> | <!-- --> | ||
| − | = | + | <span class='h3bb'>Sequence and Features</span> |
<partinfo>BBa_K3185003 SequenceAndFeatures</partinfo> | <partinfo>BBa_K3185003 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 | ||
===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K3185003 parameters</partinfo> | <partinfo>BBa_K3185003 parameters</partinfo> | ||
<!-- --> | <!-- --> | ||
Revision as of 13:43, 17 October 2019
SPYCatcher
Usage and Biology
SpyCatcher is a protein that came from the CnaB2 domain of FbaB, Streptococcus pyogenes. In a natural environment, CnaB2 domain is used for attaching to host cells. In a paper, it is partially changed and divided into two domains. (Peptide tag forming a rapid covalent bond to a protein, through engineered bacterial adhesin)
Nowadays, these two protein domains are known as SpyCatcher/SpyTag system because they bind irreversibly with a covalent bond.
In our experiment, we used the SpyCatcher/SpyTag system and designed only SpyCatcher part for assay. In addition, this has two tag or cleavage sites. First is 6×His-tag inserted in the N-terminus of SpyC for protein purification. Second is a TEV protease site because, in the paper, it was used for protein purification. (Programmable polyproteins built using twin peptide superglues) However, we didn’t use it in our experiment.
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 by using SDS-PAGE.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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