Difference between revisions of "Part:BBa K3185006"
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<partinfo>BBa_K3185006 short</partinfo> | <partinfo>BBa_K3185006 short</partinfo> | ||
==Usage and Biology== | ==Usage and Biology== | ||
− | LCI is a protein from Bacillus subtili. The paper | + | LCI is a protein from Bacillus subtili. The paper shows that it can bind to polypropylene(PP)[1]. |
− | The paper | + | The paper shows the improved variant, LCI-KR2(Y29R and G35R; variant KR-2)[2]. Its affinity is 5.4±0.5 times stronger than natural LCI. |
<br> | <br> | ||
<br> | <br> | ||
− | We used LCI-KR2 for binding protein to PP. We inserted superfolder GFP (sfGFP) whose folding interval is shortened by improving natural GFP on the N-terminus of LCI ( | + | We used LCI-KR2 for binding protein to PP. We inserted superfolder GFP (sfGFP) whose folding interval is shortened by improving natural GFP on the N-terminus of LCI (''<partinfo>BBa_BBa_I746916</partinfo>''). By doing so we wanted to do the binding assay with fluorescence. Moreover, we put SpyCatcher on N-terminus of sfGFP because we used SpyTag/SpyCatcher system to bind it to other parts. |
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− | This part has four tags. First is 6×His-tag inserted on the N-terminus of SpyC for protein purification. Second is MYC-tag inserted between sfGFP and Spy-Catcher to detect it by using the antibody. The third is a TEV protease site and we put it into two regions because it was used for protein purification in the paper | + | This part has four tags. First is 6×His-tag inserted on the N-terminus of SpyC for protein purification. Second is MYC-tag inserted between sfGFP and Spy-Catcher to detect it by using the antibody. The third is a TEV protease site and we put it into two regions because it was used for protein purification in the paper[3]. |
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− | ==References== | + | ==References== |
+ | [1](Anchor peptides: A green and versatile method for polypropylene functionalization) | ||
+ | <br> | ||
+ | [2](KnowVolution of the Polymer-Binding Peptide LCI for Improved Polypropylene Binding) | ||
+ | <br> | ||
+ | [3](Directed evolution of polypropylene and polystyrene binding peptides/Programmable polyproteams built using twin peptide superglues) | ||
<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K3185006 parameters</partinfo> | <partinfo>BBa_K3185006 parameters</partinfo> | ||
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Revision as of 11:37, 18 October 2019
SPYCatcher -> sfGFP -> LCI KR-2
Usage and Biology
LCI is a protein from Bacillus subtili. The paper shows that it can bind to polypropylene(PP)[1].
The paper shows the improved variant, LCI-KR2(Y29R and G35R; variant KR-2)[2]. Its affinity is 5.4±0.5 times stronger than natural LCI.
We used LCI-KR2 for binding protein to PP. We inserted superfolder GFP (sfGFP) whose folding interval is shortened by improving natural GFP on the N-terminus of LCI (No part name specified with partinfo tag.). By doing so we wanted to do the binding assay with fluorescence. Moreover, we put SpyCatcher on N-terminus of sfGFP because we used SpyTag/SpyCatcher system to bind it to other parts.
This part has four tags. First is 6×His-tag inserted on the N-terminus of SpyC for protein purification. Second is MYC-tag inserted between sfGFP and Spy-Catcher to detect it by using the antibody. The third is a TEV protease site and we put it into two regions because it was used for protein purification in the paper[3].
We inserted it into the site between the BamHI site on the pGEX11-a and Ndel site. We used BL21 (DE3) for gene expression. We used Ni-NTA Agarose for purification. After that, we confirmed the molecular weight of LCI by using SDS-PAGE. The result is shown below.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 1174
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Unknown
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
References
[1](Anchor peptides: A green and versatile method for polypropylene functionalization)
[2](KnowVolution of the Polymer-Binding Peptide LCI for Improved Polypropylene Binding)
[3](Directed evolution of polypropylene and polystyrene binding peptides/Programmable polyproteams built using twin peptide superglues)