BBa_K314987

BBa_K314987 Version 1

Component

This part has been discontinued.

Source:
http://parts.igem.org/Part:BBa_K314987
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


Types
DnaRegion

Roles
Coding

CDS

Sequences BBa_K314987_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
2,12
14,26
30,55
60,65
75,90
promoter feature/promoter
ribosome_entry_site feature/rbs
feature/protein CDS
stop_codon feature/stop
stop_codon feature/stop
igem#experience
None
 
igem#sampleStatus
Discontinued
igem#status
Deleted
 
synbiohub#ownedBy
user/james
 
synbiohub#ownedBy
user/myers
 
synbiohub#topLevel
BBa_K314987/1