"
Team:Hawaii/Project/Part B
From 2008.igem.org
(→Expression System for Synechocystis sp. PCC6803) |
|||
| Line 1: | Line 1: | ||
== Expression System for ''Synechocystis'' sp. PCC6803== | == Expression System for ''Synechocystis'' sp. PCC6803== | ||
| + | ==Introduction== | ||
| + | Production of proteins with clinical or industrial value can be performed by bacterial cell cultures. Once synthesized, however, problems may arise in recovering these recombinant proteins in substantial yields. Protein recovery oftentimes requires the cells to be lysed in order to recover the protein of interest, thus limiting the amount of product to a one-time yield of the total biomass that can be maintained at a given time. Production can be optimized if multiple yields can be collected over time from a living cell culture. <sup>[1]</sup> Ideally, this can be done by transporting the protein product out of the cell and collecting it from the growth medium. | ||
| - | [ | + | ==Background== |
| + | Cyanobacteria are defined by their ability to photosynthesize and can be engineered to synthesize substances of biotechnological interest. Thus cyanobacteria provide the opportunity for autotrophic production of practically any biomolecule. The ability to extract engineered biomolecules will make this bacterium a renewable, nearly self-sustaining "factory" that may revolutionize current approaches to biological engineering. The Gram-positive bacterium ''Bacillus subtilis'' has been used to produce high quality industrial enzymes and a few eukaryotic proteins via secretion into the growth medium. <sup>[2]</sup> ''Synechocystis'' sp. PCC6803 is a Gram-negative cyanobacterium that has undergone continuous study. The genome of ''Synechocystis'' has been completely sequenced <sup>[3]</sup>, making it a model organism for the exploration of secretion in Gram-negative bacteria. <sup>[4]</sup> | ||
| - | [[ | + | The major obstacle for secretion of proteins in Gram-negative bacteria is the additional outer membrane with a cell wall made of peptidoglycan murein. As a Gram-negative bacteria, ''Synechocystis'' sp. PCC6803 possess a functional periplasm between its inner and outer membranes <sup>[5]</sup>. Proteins destined for extracellular secretion must first be transported through both of these membranes and the periplasmic space. |
| + | |||
| + | Proteins are known to be secreted by bacteria via a number of different pathways. <sup>[6]</sup> The simplest way to exploit PCC6803 as a protein production factory is to take advantage of naturally existing protein secretion pathways in the organism. | ||
| + | |||
| + | Seven distinct proteins have been found to be secreted by ''Synechocystis'' sp. PCC6803 into its culture medium under normal growth conditions. <sup>[7]</sup> Analysis of the amino-terminal sequences of these proteins led to the identification of protein secretion signal polypeptides by Sergeyenko and Los. Additional studies have produced recombinant cyanobacterial strains that use PCC6803 signal peptides to secrete a foreign reporter protein, lichenase, into the culture medium. <sup>[8]</sup> | ||
| + | |||
| + | ==Objective== | ||
| + | To create BioBricks encoding signal peptides that can be combined with a protein coding sequence in order to express the protein of interest extracellularly. The ability of the signal peptide to export a protein will be tested by combining it in a BioBrick device with a fusion BioBrick part for Green Fluorescent Protein (GFP) and a BioBrick part of the nitrate-inducible ''nirA'' promoter. | ||
| + | |||
| + | ==Materials and Methods== | ||
Revision as of 23:12, 1 July 2008
Contents |
Expression System for Synechocystis sp. PCC6803
Introduction
Production of proteins with clinical or industrial value can be performed by bacterial cell cultures. Once synthesized, however, problems may arise in recovering these recombinant proteins in substantial yields. Protein recovery oftentimes requires the cells to be lysed in order to recover the protein of interest, thus limiting the amount of product to a one-time yield of the total biomass that can be maintained at a given time. Production can be optimized if multiple yields can be collected over time from a living cell culture. [1] Ideally, this can be done by transporting the protein product out of the cell and collecting it from the growth medium.
Background
Cyanobacteria are defined by their ability to photosynthesize and can be engineered to synthesize substances of biotechnological interest. Thus cyanobacteria provide the opportunity for autotrophic production of practically any biomolecule. The ability to extract engineered biomolecules will make this bacterium a renewable, nearly self-sustaining "factory" that may revolutionize current approaches to biological engineering. The Gram-positive bacterium Bacillus subtilis has been used to produce high quality industrial enzymes and a few eukaryotic proteins via secretion into the growth medium. [2] Synechocystis sp. PCC6803 is a Gram-negative cyanobacterium that has undergone continuous study. The genome of Synechocystis has been completely sequenced [3], making it a model organism for the exploration of secretion in Gram-negative bacteria. [4]
The major obstacle for secretion of proteins in Gram-negative bacteria is the additional outer membrane with a cell wall made of peptidoglycan murein. As a Gram-negative bacteria, Synechocystis sp. PCC6803 possess a functional periplasm between its inner and outer membranes [5]. Proteins destined for extracellular secretion must first be transported through both of these membranes and the periplasmic space.
Proteins are known to be secreted by bacteria via a number of different pathways. [6] The simplest way to exploit PCC6803 as a protein production factory is to take advantage of naturally existing protein secretion pathways in the organism.
Seven distinct proteins have been found to be secreted by Synechocystis sp. PCC6803 into its culture medium under normal growth conditions. [7] Analysis of the amino-terminal sequences of these proteins led to the identification of protein secretion signal polypeptides by Sergeyenko and Los. Additional studies have produced recombinant cyanobacterial strains that use PCC6803 signal peptides to secrete a foreign reporter protein, lichenase, into the culture medium. [8]
Objective
To create BioBricks encoding signal peptides that can be combined with a protein coding sequence in order to express the protein of interest extracellularly. The ability of the signal peptide to export a protein will be tested by combining it in a BioBrick device with a fusion BioBrick part for Green Fluorescent Protein (GFP) and a BioBrick part of the nitrate-inducible nirA promoter.
