Difference between revisions of "Part:BBa K128001:Experience"
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===Improvement of BBa_K128001=== | ===Improvement of BBa_K128001=== | ||
| − | The Freiburg iGEM Team 2013 wanted to use an HA-tag for cloning it into all of our versatile mammalian fusion constructs being able to detect their expression via Western blot. Therefore we searched in the parts registry and found the BBa_K128001 containing the RFC23 pre- and suffix. With this silver standard it is possible to construct in-frame-fusion parts. In contrast to the RFC25 biobrick (which is also design for functional part fusions) there is no start codon and stop codon automatically included in the prefix and suffix. To clone this HA-tag at the N-or C-terminal part of a protein it is necessary to introduce the missing start or stop codon, see Fig.1 and 2. As tags are often fused at the end (C- or N-terminal) of proteins, this HA-tag cannot that easily cloned in a functional way. We always cloned the HA-tag N-terminal to the remaining parts of our devices. In order to | + | The Freiburg iGEM Team 2013 wanted to use an HA-tag for cloning it into all of our versatile mammalian fusion constructs being able to detect their expression via Western blot. Therefore we searched in the parts registry and found the BBa_K128001 containing the RFC23 pre- and suffix. With this silver standard it is possible to construct in-frame-fusion parts. In contrast to the RFC25 biobrick (which is also design for functional part fusions) there is no start codon and stop codon automatically included in the prefix and suffix. To clone this HA-tag at the N-or C-terminal part of a protein it is necessary to introduce the missing start or stop codon, see Fig.1 and 2. As tags are often fused at the end (C- or N-terminal) of proteins, this HA-tag cannot that easily cloned in a functional way. We always cloned the HA-tag N-terminal to the remaining parts of our devices. In order to save time and efforts, we improved the consisting HA-tag BBa_K128001. Therefore we designed a new HA-tag in the RFC25 standard but with an identical sequence of amino acids. This should also facilitate cloning for other iGEM teams, see Fig.3. |
For more information about that improved HA, please take a look at BBa_K128001. | For more information about that improved HA, please take a look at BBa_K128001. | ||
Revision as of 21:04, 30 September 2013
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Improvement of BBa_K128001
The Freiburg iGEM Team 2013 wanted to use an HA-tag for cloning it into all of our versatile mammalian fusion constructs being able to detect their expression via Western blot. Therefore we searched in the parts registry and found the BBa_K128001 containing the RFC23 pre- and suffix. With this silver standard it is possible to construct in-frame-fusion parts. In contrast to the RFC25 biobrick (which is also design for functional part fusions) there is no start codon and stop codon automatically included in the prefix and suffix. To clone this HA-tag at the N-or C-terminal part of a protein it is necessary to introduce the missing start or stop codon, see Fig.1 and 2. As tags are often fused at the end (C- or N-terminal) of proteins, this HA-tag cannot that easily cloned in a functional way. We always cloned the HA-tag N-terminal to the remaining parts of our devices. In order to save time and efforts, we improved the consisting HA-tag BBa_K128001. Therefore we designed a new HA-tag in the RFC25 standard but with an identical sequence of amino acids. This should also facilitate cloning for other iGEM teams, see Fig.3.
For more information about that improved HA, please take a look at BBa_K128001.
Fig.1: BBa_K128001 with RFC23 prefix and suffix.
Fig.2: BBa_K1150016 with RFC25 prefix and suffix.
Fig.3: BBa_K1150016 theoretically fused C-terminal to a NLS with an automatic functional stop codon in the prefix.
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