Difference between revisions of "Part:BBa K3002014"

Revision as of 10:07, 13 October 2019

Mutant PETase for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of a mutant version of the PETase (B3-B4) and was built as a part of the Kaiser Collection. This part is codon-optimized for Chlamydomonas reinhardtii. The following mutations were inserted to increase activity: R280A, S238F and W159H (Joo, S. et al., 2018) (Austin, H. et al, 2018). Combined with a promoter and a terminator, this level 0 construct mediates PET degradation ability. As this part contains the introns 1-3 of RBCS2, it perfectly matches the part BBa_K3002027 (pAR promoter A1-B2), resulting in a high expression (Eichler-Stahlberg et al., 2009). To detect or purify the target protein a tag of the Kaiser Collection like BBa_K3002010 (sp20 HA-tag), BBa_K3002017 (HA-tag), BBa_K3002018 (sp20 His-tag), BBa_K3002028 (His-tag) is recommended. A secretion signal (BBa_K3002007 (cCA), BBa_K3002008 (GLE) or BBa_K3002009 (ARS)) can be added, when using the part BBa_K3002003 (pAR promoter (A1-A3)) as a promoter.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 507
Illegal PstI site found at 1480
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 507
Illegal PstI site found at 1480
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 507
Illegal PstI site found at 1480
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 507
Illegal PstI site found at 1480
Illegal NgoMIV site found at 242
Illegal NgoMIV site found at 269
1000
COMPATIBLE WITH RFC[1000]

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-This basic part contains the coding sequence of a mutant version of the PETase (B3-B4) and was built as a part of the Kaiser Collection. This part is codon-optimized for Chlamydomonas reinhardtii. The following mutations were inserted to increase activity: R280A, S238F and W159H (<a href="https://www.nature.com/articles/s41467-018-02881-1/">Joo, S. et al., 2018</a>) (<a href="https://doi.org/10.1073/pnas.1718804115">Austin, H. et al, 2018</a>). Combined with a promoter and a terminator, this level 0 construct mediates PET degradation ability. As this part contains the introns 1-3 of RBCS2, it perfectly matches the part <a href="https://parts.igem.org/Part:BBa_K3002027">BBa_K3002027</a> (pAR promoter A1-B2), resulting in a high expression (<a href="https://www.researchgate.net/publication/23762345_Strategies_to_facilitate_transgene_expression_in_Chlamydomonas_reinhardtii">Eichler-Stahlberg et al., 2009</a>). To detect or purify the target protein a tag of the Kaiser Collection like <a href="https://parts.igem.org/Part:BBa_K3002010">BBa_K3002010</a> (sp20 HA-tag), BBa_K3002017 (HA-tag), <a href="https://parts.igem.org/Part:BBa_K3002018">BBa_K3002018</a> (sp20 His-tag), <a href="https://parts.igem.org/Part:BBa_K3002028">BBa_K3002028</a> (His-tag) is recommended.
+This basic part contains the coding sequence of a mutant version of the PETase (B3-B4) and was built as a part of the Kaiser Collection. This part is codon-optimized for Chlamydomonas reinhardtii. The following mutations were inserted to increase activity: R280A, S238F and W159H (<a href="https://www.nature.com/articles/s41467-018-02881-1/">Joo, S. et al., 2018</a>) (<a href="https://doi.org/10.1073/pnas.1718804115">Austin, H. et al, 2018</a>). Combined with a promoter and a terminator, this level 0 construct mediates PET degradation ability. As this part contains the introns 1-3 of RBCS2, it perfectly matches the part <a href="https://parts.igem.org/Part:BBa_K3002027">BBa_K3002027</a> (pAR promoter A1-B2), resulting in a high expression (<a href="https://www.researchgate.net/publication/23762345_Strategies_to_facilitate_transgene_expression_in_Chlamydomonas_reinhardtii">Eichler-Stahlberg et al., 2009</a>). To detect or purify the target protein a tag of the Kaiser Collection like <a href="https://parts.igem.org/Part:BBa_K3002010">BBa_K3002010</a> (sp20 HA-tag), <a href="https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a> (HA-tag), <a href="https://parts.igem.org/Part:BBa_K3002018">BBa_K3002018</a> (sp20 His-tag), <a href="https://parts.igem.org/Part:BBa_K3002028">BBa_K3002028</a> (His-tag) is recommended.
 A secretion signal (<a href="https://parts.igem.org/Part:BBa_K3002007">BBa_K3002007</a> (cCA), <a href="https://parts.igem.org/Part:BBa_K3002008">BBa_K3002008</a> (GLE) or <a href="https://parts.igem.org/Part:BBa_K3002009">BBa_K3002009</a> (ARS)) can be added, when using the part <a href="https://parts.igem.org/Part:BBa_K3002003">BBa_K3002003</a> (pAR promoter (A1-A3)) as a promoter.