Team:Sydney Australia/Basic Part

BASIC PARTS

Our basic parts are the fundamental bricks that make up our composite parts, and include regulatory regiongs such as an efficient ribosome binding site, tags for modifying production of proteins of interest, such as affinity chromatography tags and tags for a variety of translocations within various cell types. They also contain the primary protein coding regions that we studied in this project – Human Proinsulin (BBa_K2417006)and Winsulin (BBa_K2417007).




PART NO.

 


PART NAME


SOURCE


DESCRIPTION

 

 

BBa_K2417004

 

Glycine Glycine Serine (x4) Linker Motif A

 

 

Designed

·      Linker motif used to provide spacer region

·      Used to expose affinity purification tags such as 6x His tag

·      Serine and glycine used as they are small and flexible

 

 

 

BBa_K2417006

 

 

Human Proinsulin Coding Sequence

 

 

 

Humans

·      Coding sequence for human proinsulin, sourced from NCIB

·      Consists of A, B and C chains, but C-peptide is cleaved out

·      Contains 3 disulphide bonds

·      Contains arginine residues adjacent to C-peptide to allow cleavage

 

 

BBa_K2417007

 

Winsulin - Single Chain Insulin Analogue

 

 

Designed

·      Consists of short linker peptide instead of C-peptide

·      Does not require cleavage therefore simplifies production procedures

·      Linker peptide raises pI of insulin analogue (long lasting after administration)

·      Increased thermostability (based on other single chain insulin analogues tested)

 

 

BBa_K2417008

 

6x Poly-Histidine Tag codon optimized for use in E. coli


Designed (optimized for use in Escherichia coli)

·      Codon optimized for use in E. coli

·      Used to immobilize ions on a chromatography affinity column (nickel, cobalt or copper)

·      Small, and easy to add to the ends of proteins of interest



BBa_K2417009


Extended ribosome binding site

 

 

T7 major capsid protein

·      High ribosomal recruitment efficiency

·      Sourced from the pET15b vectors

 

 

BBa_K2417010

 

 

Ecotin Periplasmic Signal Peptide

 

 

Escherichia coli

·      Small periplasmic, homodimeric protein

·      Upon fusion to N-terminus of protein will translocate protein to periplasm of E. coli

·      Periplasm is an oxidative environment ideal for protein folding, and simplifies extraction due to eliminating the need for purifying inclusion bodies from the cytoplasm

·      Periplasm has reduced proteases

 



BBa_K2417011

 

 

YncM Bacillus Secretion Peptide

 

 

Bacillus subtilis

·      Can be fused to the N-terminus of proteins to induce secretion to the surrounding media wheN proteins are expressed in Bacillus subtilis

·      Eliminates many steps in the purification process

 

 

BBa_K2417012

 

 

 

TEV Protease Cleavage Sequence

 

 

N/A

 

 

·      Sequence for which TEV protease is specific to

·      Can be added between protein of interest and peptides that require removal, such as purification tags

 

 

BBa_K2417013

 

Glycine Glycine Serine (x4) Linker

Motif B

 

 

Designed

·      Linker motif used to provide spacer region

·      Used to expose affinity purification tags such as 6x His tag

·      Serine and glycine used as they are small and flexible