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<h2 id='part-collection'><strong><span>Part Collection</span></strong></h2> | <h2 id='part-collection'><strong><span>Part Collection</span></strong></h2> | ||
− | <p><span> </span><span>This year, we | + | <p><span> </span><span>This year, we successfully obtained and expressed six manganese peroxidase (MnP) mutants as our part collection. These mutants, including MnP (E74P), MnP (E74M), MnP (D182T), MnP (S232P), MnP (E74L) and MnP(S78P), have the same function, which could all be used to degrade PE. However, they differs from each other on the stability under specific conditions. For example, MnP (E74L) has a higher enzyme activity than wild-type MnP at room temperature, while MnP (E74M) can remain stable at relatively high temperature. By carrying out protein characterization experiments, we could find the MnP mutant which has the best ability to degrade PE.</span> |
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<p><span> </span><span> For our part collection, we characterized three different fusion proteins, i.e. | <p><span> </span><span> For our part collection, we characterized three different fusion proteins, i.e. |
Revision as of 02:06, 22 October 2021
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PARTS OVERVIEW
![](https://static.igem.org/mediawiki/2021/3/38/T--CPU_CHINA--flying.png)
This year, among the 30 parts designed, 19 of them are basic parts and the rest are composite parts. All of the parts are designed on our own, and most of them have been tested to ensure their reliability.
Click here to learn more about our project to understand the purpose of each parts! Click the part number to see detailed information of the parts we design!
Basic Parts
Among the 19 basic parts designed this year by ourselves, BBa_K3853008 is our favorite one, which is the coding sequence of manganese peroxidase derived from Phanerochaete chrysosporium. The translated product of this part enables our multi-enzyme complex to degrade polyethylene efficiently. Besides, many other basic parts have also been designed to construct our project.
Favorite | Part Name | Type | Description | Designer | Length |
---|---|---|---|---|---|
BBa_K3853000 | coding | MnP | Peng Luo | 1074 | |
BBa_K3853001 | coding | AAO | Peng Luo | 1719 | |
BBa_K3853002 | coding | HFB1 | Peng Luo | 246 | |
BBa_K3853003 | coding | dCas9 | GongYu Liu | 4104 | |
BBa_K3853004 | coding | SpyTag | Peng Luo | 39 | |
BBa_K3853005 | coding | SpyCatcher | Peng Luo | 351 | |
BBa_K3853006 | DNA | dsDNA | GongYu Liu | 189 | |
BBa_K3853007 | DNA | gRNA scaffold | GongYu Liu | 80 | |
♥ | BBa_K3853008 | coding | his-tag-SpyTag-MnP | Peng Luo | 1248 |
BBa_K3853009 | coding | his-tag-SpyTag-AAO | Peng Luo | 1893 | |
BBa_K3853010 | coding | his-tag-SpyTag-HFB1 | Peng Luo | 420 | |
BBa_K3853011 | coding | dCas9-SpyCatcher | GongYu Liu | 4530 | |
BBa_K3853012 | coding | sfGFP | Peng Luo | 720 | |
BBa_K3853013 | coding | MnP(E74P) | RuYi Ma | 1074 | |
BBa_K3853014 | coding | MnP(E74M) | RuYi Ma | 1074 | |
BBa_K3853015 | coding | MnP(D182T) | RuYi Ma | 1074 | |
BBa_K3853016 | coding | MnP(S232P) | RuYi Ma | 1074 | |
BBa_K3853017 | coding | MnP(E74L) | RuYi Ma | 1074 | |
BBa_K3853018 | coding | MnP(S78P) | RuYi Ma | 1074 | |
BBa_K3853019 | coding | his-tag | Peng Luo | 18 |
Composite Parts
A series of composite parts have been designed by us this year, and most of them are used for the assembly of the PE-degrading complex in our project throughout the season.
To achieve this assembly, we designed fusion enzymes and proteins containing SpyTag and fusion deactivated CRISPR-associated protein 9 (dCas9) containing SpyCatcher, enabling the incubation-induced connection of multi-protein complexes via covalent bond formation mediated by the two Spy domains. Subsequently, these multi-protein complexes could anchor to designated positions on the designed double-stranded DNA (dsDNA) template through the guidance of dCas9-combined sgRNAs.
Our favorite composite part is BBa_K3853051 , which contains the sequence of manganese peroxidase (MnP) for polyethylene degradation, and the sequence of SpyTag for multi-domain complex formation, as well as sequences of AOX1 promoter and AOX1 terminator, enabling its efficient inducible expression in Pichia pastoris.
Favorite | Part Name | Type | Description | Designer | Length |
---|---|---|---|---|---|
BBa_K3853050 | composite | PAOX1-α-factor-sfGFP-AOX1 Terminator | Peng Luo | 2136 | |
♥ | BBa_K3853051 | composite | PAOX1-α-factor-his-tag-SpyTag-MnP-AOX1 Terminator | Peng Luo | 2664 |
BBa_K3853053 | composite | PAOX1-α-factor-his-tag-SpyTag-AAO-AOX1 Terminator | Peng Luo | 3309 | |
BBa_K3853054 | composite | R0085-C0012-B2002-his-tag-SpyTag-HFB1-B0012 | Peng Luo | 1657 | |
BBa_K3853055 | composite | R0085-C0012-B2002-his-tag-dCas9-SpyCatcher-B0012 | Peng Luo | 5758 | |
BBa_K3853056 | composite | PAOX1-α-factor-his-tag-MnP(E74P)-AOX1 Terminator | Peng Luo | 2508 | |
BBa_K3853057 | composite | PAOX1-α-factor-his-tag-MnP(E74M)-AOX1 Terminator | Peng Luo | 2508 | |
BBa_K3853058 | composite | PAOX1-α-factor-his-tag-MnP(D182T)-AOX1 Terminator | Peng Luo | 2508 | |
BBa_K3853059 | composite | PAOX1-α-factor-his-tag-MnP(S232P)-AOX1 Terminator | Peng Luo | 2508 | |
BBa_K3853060 | composite | PAOX1-α-factor-his-tag-MnP(E74L)-AOX1 Terminator | Peng Luo | 2508 | |
BBa_K3853061 | composite | PAOX1-α-factor-his-tag-MnP(S78P)-AOX1 Terminator | Peng Luo | 2508 |
Part Collection
This year, we successfully obtained and expressed six manganese peroxidase (MnP) mutants as our part collection. These mutants, including MnP (E74P), MnP (E74M), MnP (D182T), MnP (S232P), MnP (E74L) and MnP(S78P), have the same function, which could all be used to degrade PE. However, they differs from each other on the stability under specific conditions. For example, MnP (E74L) has a higher enzyme activity than wild-type MnP at room temperature, while MnP (E74M) can remain stable at relatively high temperature. By carrying out protein characterization experiments, we could find the MnP mutant which has the best ability to degrade PE.
For our part collection, we characterized three different fusion proteins, i.e. SpyTag-MnP, SpyTag-AAO, SpyTag-HFB1, and uploaded their complete and detailed characterization data to the iGEM part registry. We hope these organized information can be instructive for the design and charaterization of upcoming teams who wish to adopt these protein-nucleic-acid complex.
Peptide segment | Fusion | Part Name | Type | Description | Designer | Length |
---|---|---|---|---|---|---|
SpyTag | N-terminal | BBa_K3853051 | composite | PAOX1-α-factor-his-tag-SpyTag-MnP-AOX1 Terminator | Peng Luo | 2664 |
BBa_K3853053 | composite | PAOX1-α-factor-his-tag-SpyTag-AAO-AOX1 Terminator | Peng Luo | 3309 | ||
BBa_K3853054 | composite | PAOX1-α-factor-his-tag-SpyTag-HFB1-AOX1 Terminator | Peng Luo | 1836 |