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LuxI is one of the generated proteins in this process and functions as an AHL synthase. It converts S-adenozilmetionin to AHL which leads to higher concentration of AHL in the cell causing more LuxR molecules to become active. This is how positive feedback is formed. Expression of the ''lux''R gene is regulated by several factors such as heat shock, catabolite repression (CRP), and even LuxR itself (at very high concentration of AHL) [2]. | LuxI is one of the generated proteins in this process and functions as an AHL synthase. It converts S-adenozilmetionin to AHL which leads to higher concentration of AHL in the cell causing more LuxR molecules to become active. This is how positive feedback is formed. Expression of the ''lux''R gene is regulated by several factors such as heat shock, catabolite repression (CRP), and even LuxR itself (at very high concentration of AHL) [2]. | ||
With new tools of synthetic biology we can create devices for protein expression using parts of lux operon. We can replace | With new tools of synthetic biology we can create devices for protein expression using parts of ''lux'' operon. We can replace ''lux''I, ''lux''C, ''lux''D, ''lux''A, ''lux''B and ''lux''E genes with our gene of interest but leave the LuxR as transcriptional activator; see scheme [http://parts.igem.org/File:Luxrreceiverschematic.png here]. ''Scheme description'': Schematic LuxR receiver. Expression of ''lux''R gene is under constitutive or inducible promoter. AHL activates LuxR which is ''lux''R gene product. Active LuxR then causes a transcription of our gene of interest. RBS = ribosome binding site, T = terminator, LuxpR = LuxR promoter, PoPS-out = signal that comes out (e.g. GFP) and can be measured. | ||
Therefore AHL can be used as signaling molecule which is, as you will see in the continuation of this page, the basis of programmed pattern formation. | Therefore AHL can be used as signaling molecule which is, as you will see in the continuation of this page, the basis of programmed pattern formation. |
Revision as of 09:58, 10 January 2015
(Mitja Crček)
A synthetic multicellular system for programmed pattern formation
Subhayu Basu, Yoram Gerchman, Cynthia H. Collins, Frances H. Arnold & Ron Weiss
Nature 434, 1130-1134 (28 April 2005)
INTRODUCTION
Pattern formation is a frequently observed behavior in the living world. Many years ago scientists started to examine a pattern formation in prokaryotes and later also in eukaryotes. Here we focus on bacterial patterns because their formation has lower complexity and is therefore easier to understand. Microbiologists found that in nature, bacteria have to deal with many different environmental conditions. To increase the viability of the single bacteria, bacterial colonies are formed. To do that, bacteria had to develop complex communication pathways. Those networks are nowadays well studied and therefore synthetic biologists could start researching synthetic multicellular systems for programmed pattern formation.
Here we first explain the basics of cell-cell communication using acyl-homoserine lactone (AHL) as signal molecule so the pattern formation process will be easier to understand. We describe Tet regulatory system, lux operon, lac operon and lac repressor to explain the molecular basis and show the role of AHL.
In the second section we explain how the signaling process works in engineering cells and how the output protein is produced. We describe the molecular network in sender cells and receiver cells. The correlation between various AHL concentrations (high, medium or low) and output protein production (e.g. GFP) is shown as well as the role of repressors and activators. We show why only the receiver cells that are at appropriate distances from the sender cells can express GFP.
In the third part we focus on pattern formation. We first describe the construction of plasmids for sender and for receiver cells. We explain how bacterial strains for experiments were prepared and show simulations and experimental result of their GFP production. Illustrated with photographs we describe circular pattern formation and explain why the system works like that. Last but not least we describe the basics of ring formation dynamics and show the design of other patterns.
1 AHL signaling
Cell-cell communication is the basis to explore the pattern formation. To understand the process of communication between bacterial cells using acyl-homoserine lactone (AHL), we will first discuss the Tet regulatory system, lux operon, formation of AHL and the lac repressor.
1.1 Tet regulatory system
Tetracycline-controlled transcriptional activation is a method of inducible gene expression. Antibiotic tetracycline or its derivates (e.g. doxycycline) are used to reversibly turn the transcription of the target gene on or off. Transcription is regulated by tetracycline transactivator (tTA) protein which is formed by fusing TetR (tetracycline repressor from E.coli) with activation domain of VP16 protein (from Herpes Simplex Virus).
There are two very similar Tet regulatory systems, Tet-OFF and Tet-ON system. Both systems are based on tetracycline transactivator which activates expression. The tTA in Tet-OFF system is active in absence of tetracycline (or analogs) and is therefore capable of binding to Tet promoter which leads to transcription of target gene. When concentration of tetracycline is high, it binds to tTA and tTA is no longer capable of binding to Tet promoter. The Tet-ON system works similarly, but in the opposite way. 4 amino acid mutations of tTA allow binding of transactivator to the Tet promoter only in presence of tetracycline (or analogs). In absence of tetracycline, the tTA in Tet-ON system is unable to bind to the promoter. The simplicity of this mechanism is one of the main reasons why Tet regulatory system is commonly used in both prokaryotic and eukaryotic systems [1].
1.2 Lux operon
The lux operon encodes genes for self-regulation and was first found in Vibrio fischeri. Regulation is based on the presence/absence of AHL and crucial protein regulator is transcription activator LuxR; see Figure 1 here [2]. Figure description: Quorum-sensing model in P. fischeri using lux operon. LuxI synthesizes AHL which causes dimerization and activation of LuxR (transcriptional activator whose gene luxR is under the constitutive promoter). Active LuxR can bind to pLuxR promoter which leads to transcription of other genes in lux operon, also luxI (positive feedback). SAM = S-adenozilmetionin, ACP = acyl transfer protein, CRP = repressor protein for cAMP [2].
LuxR gene is under the constitutive promoter. Its protein product LuxR has a binding site for AHL and is inactive in absence or at very low concentrations of AHL (<10 nM). As seen in Figure 1 LuxR is activated when AHL binds and causes dimerization. Active LuxR can bind to pLuxR promoter and expression of other genes in lux operon can begin.
LuxI is one of the generated proteins in this process and functions as an AHL synthase. It converts S-adenozilmetionin to AHL which leads to higher concentration of AHL in the cell causing more LuxR molecules to become active. This is how positive feedback is formed. Expression of the luxR gene is regulated by several factors such as heat shock, catabolite repression (CRP), and even LuxR itself (at very high concentration of AHL) [2].
With new tools of synthetic biology we can create devices for protein expression using parts of lux operon. We can replace luxI, luxC, luxD, luxA, luxB and luxE genes with our gene of interest but leave the LuxR as transcriptional activator; see scheme here. Scheme description: Schematic LuxR receiver. Expression of luxR gene is under constitutive or inducible promoter. AHL activates LuxR which is luxR gene product. Active LuxR then causes a transcription of our gene of interest. RBS = ribosome binding site, T = terminator, LuxpR = LuxR promoter, PoPS-out = signal that comes out (e.g. GFP) and can be measured.
Therefore AHL can be used as signaling molecule which is, as you will see in the continuation of this page, the basis of programmed pattern formation.