Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Date created: 2010-10-02 11:00:00
Date modified: 2015-05-08 01:11:55
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| Types | DnaRegion |
| Roles | CDS Coding |
| Sequences | BBa_K314986_sequence (Version 1) |
Description
There are two main obstacles limiting natural CapD as an Anthrax therapeutic. First, natural CapD is a difficult to express dimer, requiring an auto-cleavage to activate. Second, CapD is a better transpeptidase than poly-????-D-glutamate hydrolase, limiting its Anthrax decapsulating potential. To solve the first problem, we created a circular permuted, monomeric version of CapD that is easy to express and quantify. To improve hydrolysis, we used FoldIt, a computational toolbox, to design active site mutations aimed to increase hydrolysis over transpeptidation.Notes
To increase the hydrolytic ability of CapD_CP, we made point mutations to the active site. We focused our attention on two types of mutations. First, we created point mutations that can establish hydrogen bondings to a modeled transition state of our substrate in an attempt to lower the activation energy, making hydrolysis more favorable. Second, we mutated the active site into a more open and polar area in an attempt to increase the ease with which water can enter and participate in a hydrolysis reaction.Source
To build the mutant proteins, we follow the path of the central dogma. First, we created DNA that contains our mutations. Second, we induced our transformed cells containing the desired DNA to express the mutant proteins. Lastly, we harvested the proteins by lysing open the cells and filtering out non-desired cell components.| Sequence Annotation | Location | Component / Role(s) |
| mypr rib1 capd s1 s2 BBa_K314987 mypr mypr BBa_K314988 mypr mypr mypr mypr mypr rib1 rib1 capd capd s1 s1 s2 s2 BBa_K314989 | 2,12 14,26 30,55 60,65 75,90 1,144 154,164 154,164 153,296 306,316 306,316 306,316 306,316 306,316 318,330 318,330 334,359 334,359 364,369 364,369 379,394 379,394 305,448 | engineered_region feature/BioBrick ribosome_entry_site feature/rbs CDS feature/protein feature/stop stop_codon stop_codon feature/stop feature/BioBrick engineered_region feature/promoter promoter promoter feature/promoter engineered_region feature/BioBrick promoter feature/promoter promoter feature/promoter promoter feature/promoter promoter feature/promoter feature/promoter promoter feature/rbs ribosome_entry_site ribosome_entry_site feature/rbs feature/protein CDS CDS feature/protein stop_codon feature/stop stop_codon feature/stop stop_codon feature/stop feature/stop stop_codon feature/BioBrick engineered_region |