P-RBS-CWB-

BBa_K316033 Version 1

Component

Source: http://parts.igem.org/Part:BBa_K316033

Generated By: https://synbiohub.org/public/igem/igem2sbol/1

Created by: IC 2010 Team

Date created: 2010-10-25 11:00:00

Date modified: 2015-05-08 01:11:57

Pveg-spoVG-LytC-Flexible Linker-TEV Cleavage Site-AIP-His Tag

Details

• Types: DnaRegion
• Roles: Composite; engineered_region
• Sequences: BBa_K316033_sequence (Version 1)

Description

Introduction: This part was used to link the cell wall binding domain (CWB) of LytC BBa_K316030, with the quorum sensing peptide (AIP) as well as providing a cleavage site for a protease we want to detect. This construct was built as a protease detection unit.
LytC: The part carries part of LytC on its 5??? end. This was used to ligate the linker with LytC via an internal AccI restriction site that occurs naturally in ??????B. subtilis?????? LytC sequence.
Glycin Linker: The Linker separates the CWB and the AIP and creates space for the protease to access the cleavage site; it consists of two main sections. The first six amino acids (SRGSRA) were suggested to be used specifically with LytC1 . The second section consists of a several glycin residues.
TEV Cleavage Site: This sequence forms the 3??? end of the linker and is directly attached to the 5??? end of the AIP. It is 18 amino acids (GGGGENLYFQGGKLGGGG) long and was designed to be efficiently cleaved by the TEV protease BBa_K316012, as well as being codon-optimised for expression in B. subtilis.
His-Tag: To be able to purify the protein for testing, we attached a His-Tag on our linker-AIP peptide. As it would probably interfere with recognition of the AIP by the receptor it has to be removed from the final construct.
Stop Codon: In order to end translation a double stop codon was put in place.

For more information about this part of our project please visit our wiki http://2010.igem.org/Team:Imperial_College_London/Strategy or take the tourhttp://2010.igem.org/Team:Imperial_College_London/Tour/Page_One to learn more about the project.
1 PMID: 14594841
(Yamamoto et al. 2003).

Notes

Please refer to main text

Source

LytC cell wall binding domain BBa_K316030 was extracted from ??????Bacillus subtilis?????? genome using PCR with Pfu DNA polymerase. The linker, TEV protease cleavage site and his tagged auto inducing peptide were produced by direct synthesis by mwg Eurofinshttp://www.eurofinsdna.com/home.html. The two parts were combined using AccIhttp://www.neb.com/nebecomm/products/productR0161.asp restriction endonuclease.

Components

Access	Instance	Definition
public	BBa_K316001	Pveg
public	BBa_K316043	LytC-Flex-
public	BBa_B0014	BBa_B0014

Sequence Annotations

Sequence Annotation	Location	Component / Role(s)
BBa_K316001	1,97	Pveg
BBa_K316043	106,1284	LytC-Flex-
BBa_B0014	1293,1387	BBa_B0014

Other Properties

• igem#experience: None
• igem#sampleStatus: Not in stock
• igem#status: Unavailable
• synbiohub#ownedBy: user/james
• synbiohub#ownedBy: user/myers
• synbiohub#topLevel: BBa_K316033/1

Member of these Collections

• iGEM Parts Registry