Difference between revisions of "Part:BBa K1497032"
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<span style="font-size:1.2em"><b>Usage and Biology</b></span> | <span style="font-size:1.2em"><b>Usage and Biology</b></span> | ||
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<p align="justify">The protein scaffold is an assembly platform for ligand coupled target enzymes. It was designed by the Keasling Lab in 2009 in order to improve the yield and production rate of metabolic processes. The association of target enzymes with the scaffold mimic naturally occurring catalysation cascades. In these, reaction efficiencies are optimized through the passing on of intermediates between co-located enzymes. The quick processing of intermediates can help to overcome negative production effects like unstable or toxic intermediates, metabolic bottlenecks or accumulation of undesired intermediates (Dueber et al. 2009). | <p align="justify">The protein scaffold is an assembly platform for ligand coupled target enzymes. It was designed by the Keasling Lab in 2009 in order to improve the yield and production rate of metabolic processes. The association of target enzymes with the scaffold mimic naturally occurring catalysation cascades. In these, reaction efficiencies are optimized through the passing on of intermediates between co-located enzymes. The quick processing of intermediates can help to overcome negative production effects like unstable or toxic intermediates, metabolic bottlenecks or accumulation of undesired intermediates (Dueber et al. 2009). | ||
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+ | <p align="justify">The protein scaffold consists of three different protein binding domains namely the GBD (<a href="/Part:BBa_K1497024">BBa_K1497024</a>), SH3 (<a href="/Part:BBa_K1497025">BBa_K1497025</a>) and PDZ (<a href="/Part:BBa_K1497026">BBa_K1497026</a>) domain. The domains can be linked together in any number and in any order. Therefore, a broad variety of scaffold proteins can be constructed from the initial domains depending on the application. The scaffold protein shown here consists of all domains in the order GBD<sub>1</sub>SH3<sub>1</sub>PDZ<sub>1</sub> or GSP for short. The coding sequence was optimized for the expression in <i>E. coli</i> and revised for the usage as a BioBrick. Additionally, a C-terminal His-tag was added allowing easy purification of the protein. | ||
+ | <br> <br>In this BioBrick, a cysteine residue was added directly behind the His-tag. The thiole group of the cysteine can be used for covalent crosslinking of the scaffold by reactions with maleimids and haloacetamids for example. The scaffold itself contains no other cysteine residues. Thus, coupling should be regiospecific. | ||
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<span style="font-size:1.2em"><b>Functional Parameters</b></span> | <span style="font-size:1.2em"><b>Functional Parameters</b></span> | ||
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A test experiment was performed in order to provide the basis for a successful production of the Scaff-His-Cys variant. For that purpose, the product was examined by PAA gel electrophoresis (see figure 3). | A test experiment was performed in order to provide the basis for a successful production of the Scaff-His-Cys variant. For that purpose, the product was examined by PAA gel electrophoresis (see figure 3). | ||
Onto the gel, next to the marker, a sample of the pre-culture, the cells prior, and several hours after induction were applied. A clear band was visible at the height above 30 kDa. The scaffold with His-tag has a size of 31.17 kDa. As a consequence, the band appearing after induction can be considered as the correct protein. | Onto the gel, next to the marker, a sample of the pre-culture, the cells prior, and several hours after induction were applied. A clear band was visible at the height above 30 kDa. The scaffold with His-tag has a size of 31.17 kDa. As a consequence, the band appearing after induction can be considered as the correct protein. | ||
− | Due to the presence of the scaffold protein in the lane of the pre-culture, it can be concluded that the inducible promoter is not entirely locked. A clear overproduction of the scaffold protein is only reached after an incubation time of four hours. | + | Due to the presence of the scaffold protein in the lane of the pre-culture (VK), it can be concluded that the inducible promoter is not entirely locked. A clear overproduction of the scaffold protein is only reached after an incubation time of four hours. |
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The construction of the scaffold GSP-His-Cys is nearly identical to the scaffold GSP-His | The construction of the scaffold GSP-His-Cys is nearly identical to the scaffold GSP-His |
Revision as of 19:49, 15 October 2014
Scaffold (G-S-P-His-Cys)
Usage and Biology
Assembly Compatibility:
References
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