A synthetic multicellular system for programmed pattern formation - Revision history

MitjaCrcek at 18:05, 10 January 2015

2015-01-10T18:05:25Z

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	==~~'''~~References~~'''~~==		==References==

	[http://www.sciencedirect.com/science/article/pii/S1084952102000186] Z. Zhu, T. Zheng, C. G. Lee, R. J. Homer, in J. A. Elias, „Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling“, Semin. Cell Dev. Biol., Vol. 13, No. 2, pp. 121–128, apr 2002.		[http://www.sciencedirect.com/science/article/pii/S1084952102000186] Z. Zhu, T. Zheng, C. G. Lee, R. J. Homer, in J. A. Elias, „Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling“, Semin. Cell Dev. Biol., Vol. 13, No. 2, pp. 121–128, apr 2002.

MitjaCrcek at 16:58, 10 January 2015

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	=='''Formation of other patterns'''==		=='''Formation of other patterns'''==

	The final pattern and dynamics depend not only on the sender-receiver network but also on the arrangement of sender cells. It is possible to create many different patterns by placing multiple sender disks in various configurations. The number of sender cells (sender disks), their density and the distance between them lead to different AHL gradients reflected in different intricate patterns formation (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F5.html Figure 5]). ''Figure description'': Formation of different patterns. a, simulation of cell behavior on solid media with two sender cells on determined distance; formation of an ellipse. b-d, experimental results showing various patterns formed. Sender cells were expressing DsRed-Express and receiver cells GFP. Patterns are based on the placement and different concentration of sender cells. b, two sender disks; ellipse. c, three sender disks; heart. d, four sender disks; clover ~~[4]~~.		The final pattern and dynamics depend not only on the sender-receiver network but also on the arrangement of sender cells. It is possible to create many different patterns by placing multiple sender disks in various configurations. The number of sender cells (sender disks), their density and the distance between them lead to different AHL gradients reflected in different intricate patterns formation (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F5.html Figure 5] [4]). ''Figure description'': Formation of different patterns. a, simulation of cell behavior on solid media with two sender cells on determined distance; formation of an ellipse. b-d, experimental results showing various patterns formed. Sender cells were expressing DsRed-Express and receiver cells GFP. Patterns are based on the placement and different concentration of sender cells. b, two sender disks; ellipse. c, three sender disks; heart. d, four sender disks; clover.

MitjaCrcek at 16:53, 10 January 2015

2015-01-10T16:53:07Z

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	In agreement with model predictions (Figure 2a) the dosage responses of all three HD strains (containing only pHD{x}) showed reverse correlation to AHL concentrations (see Figure 2b). The only but crucial difference between them is in their sensitivities. Note that all experiments were done with transformed E.coli strain DH5α. For more details of experiment conditions see Methods in [4].		In agreement with model predictions (Figure 2a) the dosage responses of all three HD strains (containing only pHD{x}) showed reverse correlation to AHL concentrations (see Figure 2b). The only but crucial difference between them is in their sensitivities. Note that all experiments were done with transformed E.coli strain DH5α. For more details of experiment conditions see Methods in [4].

	To reach the non-monotonic response to AHL, it is required to use both vectors, high-detect and low-detect plasmid (Figure 2c and 2d). By combining the pLD with each of the pHD{x}, they obtained three different strains (named BD1, BD2 and BD3 in [4]). Those BD strains showed predicted non-monotonic response to different AHL concentrations with different thresholds (see Figure 2d). Results also correlated well with model predictions (see Figure 2c).		To reach the non-monotonic response to AHL, it is required to use both vectors, high-detect and low-detect plasmid (Figure 2c and 2d). By combining the pLD with each of the pHD{x}, they obtained three different strains (named BD1, BD2 and BD3 in [4]). Those BD (band-detect) strains showed predicted non-monotonic response to different AHL concentrations with different thresholds (see Figure 2d). Results also correlated well with model predictions (see Figure 2c).

	===Circular pattern formation===		===Circular pattern formation===

MitjaCrcek at 13:30, 10 January 2015

2015-01-10T13:30:31Z

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	The ''lac'' operon actually uses a two-part control mechanism of expression. In the presence of glucose, the catabolite activator protein remains inactive so the enzymes cannot be produced. That shuts down lactose permease to prevent transport of lactose into the cell because it does not need the lactose from the media.		The ''lac'' operon actually uses a two-part control mechanism of expression. In the presence of glucose, the catabolite activator protein remains inactive so the enzymes cannot be produced. That shuts down lactose permease to prevent transport of lactose into the cell because it does not need the lactose from the media.

	The second control mechanism is the regulatory response to lactose which is regulated by LacI. In the absence of lactose, the lac repressor binds to ''lac'' operator and therefore halts production of the enzymes encoded by the ''lac'' operon. LacI gene is under a constitutive promoter so the regulation of ''lac'' operon is constant. When there is a high concentration of lactose in the cell, lactose is converted into allolactose which inhibits the lac repressor's DNA binding ability. This leads to transcriptional activation of the ''lac'' operon ~~[3]. See~~ figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_Operon.svg here] [3]. ''Figure description'': Regulation of the ''lac'' operon. In absence of lactose, LacI binds to ''lac'' operator which obstructs the RNA polymerase from binding to the promoter (top). This results in very low levels of mRNA encoding LacZ and LacY. Once present in the cell, lactose can convert to allolactose which binds to LacI. An allosteric change in shape of LacI result as inability of binding of repressor to operator region (bottom). 1: RNA polymerase, 2: repressor, 3: promoter, 4: operator, 5: allolactose, 6: ''lac''Z, 7: ''lac''Y, 8: ''lac''A [3].		The second control mechanism is the regulatory response to lactose which is regulated by LacI. In the absence of lactose, the lac repressor binds to ''lac'' operator and therefore halts production of the enzymes encoded by the ''lac'' operon. LacI gene is under a constitutive promoter so the regulation of ''lac'' operon is constant. When there is a high concentration of lactose in the cell, lactose is converted into allolactose which inhibits the lac repressor's DNA binding ability. This leads to transcriptional activation of the ''lac'' operon; see figure [http://en.wikipedia.org/wiki/Lac_operon#mediaviewer/File:Lac_Operon.svg here] [3]. ''Figure description'': Regulation of the ''lac'' operon. In absence of lactose, LacI binds to ''lac'' operator which obstructs the RNA polymerase from binding to the promoter (top). This results in very low levels of mRNA encoding LacZ and LacY. Once present in the cell, lactose can convert to allolactose which binds to LacI. An allosteric change in shape of LacI result as inability of binding of repressor to operator region (bottom). 1: RNA polymerase, 2: repressor, 3: promoter, 4: operator, 5: allolactose, 6: ''lac''Z, 7: ''lac''Y, 8: ''lac''A [3].

	All the structural genes can be replaced with our gene of interest so we obtain a mechanism which is easy to regulate and can produce the desired protein (e.g. GFP). Expression of ''lac''I gene can be regulated with activators or repressors such as lambda repressor (cI). If we place the gene encoding for cI under the control of constitutive promoter (like pLuxR) we can control the expression of LacI and therefore the production of desired protein.		All the structural genes can be replaced with our gene of interest so we obtain a mechanism which is easy to regulate and can produce the desired protein (e.g. GFP). Expression of ''lac''I gene can be regulated with activators or repressors such as lambda repressor (cI). If we place the gene encoding for cI under the control of constitutive promoter (like pLuxR) we can control the expression of LacI and therefore the production of desired protein.

MitjaCrcek at 13:23, 10 January 2015

2015-01-10T13:23:02Z

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	=='''Signaling process and output protein production'''==		=='''Signaling process and output protein production'''==

	[http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1a] in the [http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html origin article] shows us the design of the synthetic bacterial multicellular system for a signaling process that can lead to pattern formation [4]. We can see that only receiver cells at intermediate distances from sender cells express the output protein. The signaling process begins when tetracycline (or analog) is added to the sender cells which use Tet-ON regulatory system. Tetracycline binds to TetR (which is a positive regulator) and that has an impact on the expression of LuxI. LuxI is an enzyme that synthesizes AHL, which diffuses through the cell membrane and forms a chemical gradient around the senders; see Figure 1a. ''Figure description'': A signaling process and GFP production. a, the correlation between various AHL concentrations (high, medium or low) and expression of cI, LacIM1, LacI and GFP. b, approximation of the AHL gradient as a function of the distance from the sender cells. c, signaling process. Only the receiver cells that are at appropriate distances from the sender cells can express the GFP [4].		[http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1a] in the [http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html origin article] shows us the design of the synthetic bacterial multicellular system for a signaling process that can lead to pattern formation [4]. ''Figure description'': A signaling process and GFP production. a, the correlation between various AHL concentrations (high, medium or low) and expression of cI, LacIM1, LacI and GFP. b, approximation of the AHL gradient as a function of the distance from the sender cells. c, signaling process. Only the receiver cells that are at appropriate distances from the sender cells can express the GFP [4].

			We can see that only receiver cells at intermediate distances from sender cells express the output protein. The signaling process begins when tetracycline (or analog) is added to the sender cells which use Tet-ON regulatory system. Tetracycline binds to TetR (which is a positive regulator) and that has an impact on the expression of LuxI. LuxI is an enzyme that synthesizes AHL, which diffuses through the cell membrane and forms a chemical gradient around the senders; see Figure 1a.

	AHL in media can easily diffuse into nearby receiver cells which use the Lux receiver, a part of ''lux'' operon. AHL binds to LuxR, a positive AHL-dependent transcriptional regulator, which activates the expression of two regulatory proteins, lambda repressor (cI) and modified Lac repressor - LacIM1. cI is a strong repressor from bacteriophage lambda and represses the expression of wild type LacI, which is under the constitutive promoter. LacIM1 is a product of a codon-modified ''lac''I and has (in the receiver cells) the same role as LacI - to repress the expression of the output protein (in this case the green fluorescent protein - GFP).		AHL in media can easily diffuse into nearby receiver cells which use the Lux receiver, a part of ''lux'' operon. AHL binds to LuxR, a positive AHL-dependent transcriptional regulator, which activates the expression of two regulatory proteins, lambda repressor (cI) and modified Lac repressor - LacIM1. cI is a strong repressor from bacteriophage lambda and represses the expression of wild type LacI, which is under the constitutive promoter. LacIM1 is a product of a codon-modified ''lac''I and has (in the receiver cells) the same role as LacI - to repress the expression of the output protein (in this case the green fluorescent protein - GFP).

MitjaCrcek at 13:20, 10 January 2015

2015-01-10T13:20:24Z

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	=='''Signaling process and output protein production'''==		=='''Signaling process and output protein production'''==

	[http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1a] in the [http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html origin article] shows us the design of the synthetic bacterial multicellular system for a signaling process that can lead to pattern formation [4]. We can see that only receiver cells at intermediate distances from sender cells express the output protein. The signaling process begins when tetracycline (or analog) is added to the sender cells which use Tet-ON regulatory system. Tetracycline binds to TetR (which is a positive regulator) and that has an impact on the expression of LuxI. LuxI is an enzyme that synthesizes AHL, which diffuses through the cell membrane and forms a chemical gradient around the senders (see Figure 1a).		[http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1a] in the [http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html origin article] shows us the design of the synthetic bacterial multicellular system for a signaling process that can lead to pattern formation [4]. We can see that only receiver cells at intermediate distances from sender cells express the output protein. The signaling process begins when tetracycline (or analog) is added to the sender cells which use Tet-ON regulatory system. Tetracycline binds to TetR (which is a positive regulator) and that has an impact on the expression of LuxI. LuxI is an enzyme that synthesizes AHL, which diffuses through the cell membrane and forms a chemical gradient around the senders; see Figure 1a. ''Figure description'': A signaling process and GFP production. a, the correlation between various AHL concentrations (high, medium or low) and expression of cI, LacIM1, LacI and GFP. b, approximation of the AHL gradient as a function of the distance from the sender cells. c, signaling process. Only the receiver cells that are at appropriate distances from the sender cells can express the GFP [4].

	AHL in media can easily diffuse into nearby receiver cells which use the Lux receiver, a part of ''lux'' operon. AHL binds to LuxR, a positive AHL-dependent transcriptional regulator, which activates the expression of two regulatory proteins, lambda repressor (cI) and modified Lac repressor - LacIM1. cI is a strong repressor from bacteriophage lambda and represses the expression of wild type LacI, which is under the constitutive promoter. LacIM1 is a product of a codon-modified ''lac''I and has (in the receiver cells) the same role as LacI - to repress the expression of the output protein (in this case the green fluorescent protein - GFP).		AHL in media can easily diffuse into nearby receiver cells which use the Lux receiver, a part of ''lux'' operon. AHL binds to LuxR, a positive AHL-dependent transcriptional regulator, which activates the expression of two regulatory proteins, lambda repressor (cI) and modified Lac repressor - LacIM1. cI is a strong repressor from bacteriophage lambda and represses the expression of wild type LacI, which is under the constitutive promoter. LacIM1 is a product of a codon-modified ''lac''I and has (in the receiver cells) the same role as LacI - to repress the expression of the output protein (in this case the green fluorescent protein - GFP).
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	At intermediate distances from the sender cells, intermediate AHL concentrations result in moderate levels of LacIM1 and cI. Because the repression efficiency of LacIM1 is significantly lower than that of cI, cI effectively shuts of the expression of LacI while the threshold concentration of LacIM1 required to repress GFP production is not achieved. This difference between repression efficiencies of LacIM1 and cI affords bacteria the non-monotonic response to AHL dosages.		At intermediate distances from the sender cells, intermediate AHL concentrations result in moderate levels of LacIM1 and cI. Because the repression efficiency of LacIM1 is significantly lower than that of cI, cI effectively shuts of the expression of LacI while the threshold concentration of LacIM1 required to repress GFP production is not achieved. This difference between repression efficiencies of LacIM1 and cI affords bacteria the non-monotonic response to AHL dosages.

	''Figure 1a description'': A signaling process and GFP production. a, the correlation between various AHL concentrations (high, medium or low) and expression of cI, LacIM1, LacI and GFP. b, approximation of the AHL gradient as a function of the distance from the sender cells. c, signaling process. Only the receiver cells that are at appropriate distances from the sender cells can express the GFP [4].

MitjaCrcek at 13:14, 10 January 2015

2015-01-10T13:14:45Z

MitjaCrcek at 13:03, 10 January 2015

2015-01-10T13:03:36Z

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	=='''Signaling process and output protein production'''==		=='''Signaling process and output protein production'''==

	[http://www.nature.com~~.nukweb.nuk.uni-lj.si~~/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1a] in the [http://www.nature.com~~.nukweb.nuk.uni-lj.si~~/nature/journal/v434/n7037/full/nature03461.html origin article] shows us the design of the synthetic bacterial multicellular system for a signaling process that can lead to pattern formation [4]. We can see that only receiver cells at intermediate distances from sender cells express the output protein. The signaling process begins when tetracycline (or analog) is added to the sender cells which use Tet-ON regulatory system. Tetracycline binds to TetR (which is a positive regulator) and that has an impact on the expression of LuxI. LuxI is an enzyme that synthesizes AHL, which diffuses through the cell membrane and forms a chemical gradient around the senders (see Figure 1a).		[http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1a] in the [http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html origin article] shows us the design of the synthetic bacterial multicellular system for a signaling process that can lead to pattern formation [4]. We can see that only receiver cells at intermediate distances from sender cells express the output protein. The signaling process begins when tetracycline (or analog) is added to the sender cells which use Tet-ON regulatory system. Tetracycline binds to TetR (which is a positive regulator) and that has an impact on the expression of LuxI. LuxI is an enzyme that synthesizes AHL, which diffuses through the cell membrane and forms a chemical gradient around the senders (see Figure 1a).

	AHL in media can easily diffuse into nearby receiver cells which use the Lux receiver, a part of ''lux'' operon. AHL binds to LuxR, a positive AHL-dependent transcriptional regulator, which activates the expression of two regulatory proteins, lambda repressor (cI) and modified Lac repressor - LacIM1. cI is a strong repressor from bacteriophage lambda and represses the expression of wild type LacI, which is under the constitutive promoter. LacIM1 is a product of a codon-modified ''lac''I and has (in the receiver cells) the same role as LacI - to repress the expression of the output protein (in this case the green fluorescent protein - GFP).		AHL in media can easily diffuse into nearby receiver cells which use the Lux receiver, a part of ''lux'' operon. AHL binds to LuxR, a positive AHL-dependent transcriptional regulator, which activates the expression of two regulatory proteins, lambda repressor (cI) and modified Lac repressor - LacIM1. cI is a strong repressor from bacteriophage lambda and represses the expression of wild type LacI, which is under the constitutive promoter. LacIM1 is a product of a codon-modified ''lac''I and has (in the receiver cells) the same role as LacI - to repress the expression of the output protein (in this case the green fluorescent protein - GFP).

MitjaCrcek at 13:01, 10 January 2015

2015-01-10T13:01:00Z

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	[http://www.nature.com~~.nukweb.nuk.uni-lj.si~~/nature/journal/v434/n7037/pdf/nature03461.pdf A synthetic multicellular system for programmed pattern formation]		[http://www.nature.com/nature/journal/v434/n7037/pdf/nature03461.pdf A synthetic multicellular system for programmed pattern formation]

	Subhayu Basu, Yoram Gerchman, Cynthia H. Collins, Frances H. Arnold & Ron Weiss		Subhayu Basu, Yoram Gerchman, Cynthia H. Collins, Frances H. Arnold & Ron Weiss

MitjaCrcek at 12:50, 10 January 2015

2015-01-10T12:50:46Z

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	[http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/full/nature03461.html] S. Basu, Y. Gerchman, C. H. Collins, F. H. Arnold, in R. Weiss, „A synthetic multicellular system for programmed pattern formation“, Nature, Vol. 434, No. 7037, pp. 1130–1134, apr 2005.		[http://www.nature.com.nukweb.nuk.uni-lj.si/nature/journal/v434/n7037/full/nature03461.html] S. Basu, Y. Gerchman, C. H. Collins, F. H. Arnold, in R. Weiss, „A synthetic multicellular system for programmed pattern formation“, Nature, Vol. 434, No. 7037, pp. 1130–1134, apr 2005.

	[http://~~download~~.springer.com.nukweb.nuk.uni-lj.si/~~static~~/~~pdf/256/art%253A10~~.1007~~%252Fs10126~~-001-0081-7~~.pdf?auth66=1420894086_cacdf94c6c231719ca3ef3a53b6096e8&ext=.pdf~~] N.-S. Xia, W.-X. Luo, J. Zhang, X.-Y. Xie, H.-J. Yang, S.-W. Li, M. Chen, in M.-H. Ng, „Bioluminescence of Aequorea macrodactyla, a Common Jellyfish Species in the East China Sea“, Mar. Biotechnol., Vol. 4, No. 2, pp. 155–162, mar 2002.		[http://link.springer.com.nukweb.nuk.uni-lj.si/article/10.1007/s10126-001-0081-7] N.-S. Xia, W.-X. Luo, J. Zhang, X.-Y. Xie, H.-J. Yang, S.-W. Li, M. Chen, in M.-H. Ng, „Bioluminescence of Aequorea macrodactyla, a Common Jellyfish Species in the East China Sea“, Mar. Biotechnol., Vol. 4, No. 2, pp. 155–162, mar 2002.

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	===Plasmids for sender and receiver cells===		===Plasmids for sender and receiver cells===

	To achieve the cell behavior described previously, vectors that contain a certain bio-bricks should be designed. Scientist from Princeton University and California Institute of Technology engineered vectors for sender and receiver cells and used them to reach a pattern formation in bacteria [4]. As you can see in [http://www.nature.com~~.nukweb.nuk.uni-lj.si~~/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1b], sender plasmid (pSND) contains ''lux''I gene, which is under the control of Tet promoter. For the purpose of the experiment, cells containing Tet regulatory system were transformed with pSND, so they could express LuxI. To reach a specific expression of GFP they engineered two different vectors for receiver cells and named them high-detect (pHD{x}) and low-detect (pLD) plasmids (we will explain later why there are two plasmids).		To achieve the cell behavior described previously, vectors that contain a certain bio-bricks should be designed. Scientist from Princeton University and California Institute of Technology engineered vectors for sender and receiver cells and used them to reach a pattern formation in bacteria [4]. As you can see in [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1b], sender plasmid (pSND) contains ''lux''I gene, which is under the control of Tet promoter. For the purpose of the experiment, cells containing Tet regulatory system were transformed with pSND, so they could express LuxI. To reach a specific expression of GFP they engineered two different vectors for receiver cells and named them high-detect (pHD{x}) and low-detect (pLD) plasmids (we will explain later why there are two plasmids).

	High-detect plasmid (pHD{x} - see [http://www.nature.com~~.nukweb.nuk.uni-lj.si~~/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1c]) contains ''lac''IM1, ''lux''R and ''gfp'' genes. They engineered three different high-detect strains (HD1, HD2 and HD3) to achieve the best pattern formation process. The HD1 strain contains a hypersensitive LuxR mutant, HD2 incorporates the wild-type LuxR, and HD3 strain expresses LuxR from a reduced-copy-number plasmid.		High-detect plasmid (pHD{x} - see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1c]) contains ''lac''IM1, ''lux''R and ''gfp'' genes. They engineered three different high-detect strains (HD1, HD2 and HD3) to achieve the best pattern formation process. The HD1 strain contains a hypersensitive LuxR mutant, HD2 incorporates the wild-type LuxR, and HD3 strain expresses LuxR from a reduced-copy-number plasmid.
	Low-detect plasmid (pLD - see [http://www.nature.com~~.nukweb.nuk.uni-lj.si~~/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1d]) contains ''c''I and ''lac''I genes and is crucial for non-monotonic response to AHL as you will see later.		Low-detect plasmid (pLD - see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F1.html Figure 1d]) contains ''c''I and ''lac''I genes and is crucial for non-monotonic response to AHL as you will see later.

	''Figure 1b, 1c and 1d description'': Plasmids for pattern formation. a, plasmid map for sender cells. b, high-detect plasmid. c, low-detect plasmid. Three versions of the high-detect plasmid with different sensitivities to AHL were constructed (regions of mutation are underlined: pHD1, LuxR; pHD2, wild-type; pHD3, ColE1) [4].		''Figure 1b, 1c and 1d description'': Plasmids for pattern formation. a, plasmid map for sender cells. b, high-detect plasmid. c, low-detect plasmid. Three versions of the high-detect plasmid with different sensitivities to AHL were constructed (regions of mutation are underlined: pHD1, LuxR; pHD2, wild-type; pHD3, ColE1) [4].
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	===Simulated and experimental behavior of engineered bacterial strains===		===Simulated and experimental behavior of engineered bacterial strains===

	Simulations and experimental results show how prepared bacterial strains behave in different AHL concentration (see [http://www.nature.com~~.nukweb.nuk.uni-lj.si~~/nature/journal/v434/n7037/fig_tab/nature03461_F2.html Figure 2]) [4]. ''Figure description'': Simulated and experimental liquid-phase behavior of transformed cells. a, b, simulations (a) and experimental results (b) of the AHL dosage response for HD1 strain (hypersensitive LuxR, blue), HD2 strain (wild-type LuxR, red) and HD3 (a reduced-copy-number plasmid, black). c, d, simulations (c) and experimental results (d) of three strains BD1 (blue), BD2 (red) and BD3 (black), all three consist of the same pLD plasmid and different pHD{x} plasmids (pHD1, pHD2, pHD3, respectively).		Simulations and experimental results show how prepared bacterial strains behave in different AHL concentration (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F2.html Figure 2]) [4]. ''Figure description'': Simulated and experimental liquid-phase behavior of transformed cells. a, b, simulations (a) and experimental results (b) of the AHL dosage response for HD1 strain (hypersensitive LuxR, blue), HD2 strain (wild-type LuxR, red) and HD3 (a reduced-copy-number plasmid, black). c, d, simulations (c) and experimental results (d) of three strains BD1 (blue), BD2 (red) and BD3 (black), all three consist of the same pLD plasmid and different pHD{x} plasmids (pHD1, pHD2, pHD3, respectively).

	In agreement with model predictions (Figure 2a) the dosage responses of all three HD strains (containing only pHD{x}) showed reverse correlation to AHL concentrations (see Figure 2b). The only but crucial difference between them is in their sensitivities. Note that all experiments were done with transformed E.coli strain DH5α. For more details of experiment conditions see Methods in [4].		In agreement with model predictions (Figure 2a) the dosage responses of all three HD strains (containing only pHD{x}) showed reverse correlation to AHL concentrations (see Figure 2b). The only but crucial difference between them is in their sensitivities. Note that all experiments were done with transformed E.coli strain DH5α. For more details of experiment conditions see Methods in [4].
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	Experiments shows the formation of such pattern on a plate with BD3 or BD1 cells (with ''gfp'' gene) and BD2-Red cells (similar to BD2 with ''dsRed-Express'' replacing ''gfp'' gene).		Experiments shows the formation of such pattern on a plate with BD3 or BD1 cells (with ''gfp'' gene) and BD2-Red cells (similar to BD2 with ''dsRed-Express'' replacing ''gfp'' gene).
	See [http://www.nature.com.~~nukweb.nuk.uni-lj.si~~/nature/journal/v434/n7037/fig_tab/nature03461_F3.html Figure 3] [4]. ''Figure description'': Experimental solid-phase behavior of described networks. a, photo of agar plate with the sender disk in the middle. b, bullseye pattern with sender cells in the middle, inner green fluorescent ring (made by BD3 cells) and outer red fluorescent ring (made by BD2-Red cells). c, another bullseye pattern made by mixture of BD1 and BD2-Red cells. Scale bar; 5 mm.		See [http://www.nature.com./nature/journal/v434/n7037/fig_tab/nature03461_F3.html Figure 3] [4]. ''Figure description'': Experimental solid-phase behavior of described networks. a, photo of agar plate with the sender disk in the middle. b, bullseye pattern with sender cells in the middle, inner green fluorescent ring (made by BD3 cells) and outer red fluorescent ring (made by BD2-Red cells). c, another bullseye pattern made by mixture of BD1 and BD2-Red cells. Scale bar; 5 mm.

	First, two different bacterial strains (BD3/BD1 and BD2-Red) were spread evenly on top of an agarose plate. Then, a disc containing sender cells, which express CFP (cyan fluorescent protein; GFP-like protein from jellyfish ''Aequorea macrodactyla'' [5]), was placed in the middle of the plate (Figure 3a). Those plates were incubated at 37 °C overnight. Bullseye patterns were captured with a fluorescence microscope. As seen in Figure 3b (BD3/BD2-Red experiment), BD3 cells formed a green fluorescent ring near the sender disk, whereas BD2-Red cells formed a ring further from sender cells. In the middle we can see a cyan fluorescent ring, created by sender cells, but no red or green fluorescent color. This phenomenon is the consequence of different sensitivities of BD strains to AHL (see the text above and Figure 2).		First, two different bacterial strains (BD3/BD1 and BD2-Red) were spread evenly on top of an agarose plate. Then, a disc containing sender cells, which express CFP (cyan fluorescent protein; GFP-like protein from jellyfish ''Aequorea macrodactyla'' [5]), was placed in the middle of the plate (Figure 3a). Those plates were incubated at 37 °C overnight. Bullseye patterns were captured with a fluorescence microscope. As seen in Figure 3b (BD3/BD2-Red experiment), BD3 cells formed a green fluorescent ring near the sender disk, whereas BD2-Red cells formed a ring further from sender cells. In the middle we can see a cyan fluorescent ring, created by sender cells, but no red or green fluorescent color. This phenomenon is the consequence of different sensitivities of BD strains to AHL (see the text above and Figure 2).
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	=='''Ring formation dynamics'''==		=='''Ring formation dynamics'''==

	Understanding the dynamic behavior of the system is essential for predicting pattern formation. The ring formation activity of BD2 cells was measured over the course of 36 hours to study the system dynamics. Fluorescence was recorded every 90 minutes for a rectangular region protruding from the sender disk (see [http://www.nature.com~~.nukweb.nuk.uni-lj.si~~/nature/journal/v434/n7037/fig_tab/nature03461_F4.html Figure 4a]) [4]. ''Figure description'': Results show the time-evolution of fluorescence (of GFP) for cells as a function of the distance from the sender cells. Dark blue: low fluorescence, yellow and green: intermediate fluorescence, red: high fluorescence.		Understanding the dynamic behavior of the system is essential for predicting pattern formation. The ring formation activity of BD2 cells was measured over the course of 36 hours to study the system dynamics. Fluorescence was recorded every 90 minutes for a rectangular region protruding from the sender disk (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F4.html Figure 4a]) [4]. ''Figure description'': Results show the time-evolution of fluorescence (of GFP) for cells as a function of the distance from the sender cells. Dark blue: low fluorescence, yellow and green: intermediate fluorescence, red: high fluorescence.

	For the first 15 hours no fluorescence was observed. Later, as we can see in Figure 4a, low levels of fluorescence (brighter blue and green color) emerged about 10 mm from the senders. Fluorescence was then significantly increased between 5 and 18 mm from the sender cells reaching a steady-state maximum at 10 mm.		For the first 15 hours no fluorescence was observed. Later, as we can see in Figure 4a, low levels of fluorescence (brighter blue and green color) emerged about 10 mm from the senders. Fluorescence was then significantly increased between 5 and 18 mm from the sender cells reaching a steady-state maximum at 10 mm.
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	=='''Formation of other patterns'''==		=='''Formation of other patterns'''==

	The final pattern and dynamics depend not only on the sender-receiver network but also on the arrangement of sender cells. It is possible to create many different patterns by placing multiple sender disks in various configurations. The number of sender cells (sender disks), their density and the distance between them lead to different AHL gradients reflected in different intricate patterns formation (see [http://www.nature.com~~.nukweb.nuk.uni-lj.si~~/nature/journal/v434/n7037/fig_tab/nature03461_F5.html Figure 5]). ''Figure description'': Formation of different patterns. a, simulation of cell behavior on solid media with two sender cells on determined distance; formation of an ellipse. b-d, experimental results showing various patterns formed. Sender cells were expressing DsRed-Express and receiver cells GFP. Patterns are based on the placement and different concentration of sender cells. b, two sender disks; ellipse. c, three sender disks; heart. d, four sender disks; clover [4].		The final pattern and dynamics depend not only on the sender-receiver network but also on the arrangement of sender cells. It is possible to create many different patterns by placing multiple sender disks in various configurations. The number of sender cells (sender disks), their density and the distance between them lead to different AHL gradients reflected in different intricate patterns formation (see [http://www.nature.com/nature/journal/v434/n7037/fig_tab/nature03461_F5.html Figure 5]). ''Figure description'': Formation of different patterns. a, simulation of cell behavior on solid media with two sender cells on determined distance; formation of an ellipse. b-d, experimental results showing various patterns formed. Sender cells were expressing DsRed-Express and receiver cells GFP. Patterns are based on the placement and different concentration of sender cells. b, two sender disks; ellipse. c, three sender disks; heart. d, four sender disks; clover [4].


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	=='''References'''==		=='''References'''==

	[http://www.sciencedirect.com~~.nukweb.nuk.uni-lj.si~~/science/article/pii/S1084952102000186] Z. Zhu, T. Zheng, C. G. Lee, R. J. Homer, in J. A. Elias, „Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling“, Semin. Cell Dev. Biol., Vol. 13, No. 2, pp. 121–128, apr 2002.		[http://www.sciencedirect.com/science/article/pii/S1084952102000186] Z. Zhu, T. Zheng, C. G. Lee, R. J. Homer, in J. A. Elias, „Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling“, Semin. Cell Dev. Biol., Vol. 13, No. 2, pp. 121–128, apr 2002.

	[http://mmbr.asm.org/content/67/4/574.full.pdf+html] J. E. González in M. M. Marketon, „Quorum Sensing in Nitrogen-Fixing Rhizobia“, Microbiol. Mol. Biol. Rev., Vol. 67, No. 4, pp. 574–592, jan 2003.		[http://mmbr.asm.org/content/67/4/574.full.pdf+html] J. E. González in M. M. Marketon, „Quorum Sensing in Nitrogen-Fixing Rhizobia“, Microbiol. Mol. Biol. Rev., Vol. 67, No. 4, pp. 574–592, jan 2003.
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	[http://en.wikipedia.org/wiki/Lac_operon] „lac operon“, Wikipedia, the free encyclopedia. 20-nov-2014.		[http://en.wikipedia.org/wiki/Lac_operon] „lac operon“, Wikipedia, the free encyclopedia. 20-nov-2014.

	[http://www.nature.com~~.nukweb.nuk.uni-lj.si~~/nature/journal/v434/n7037/full/nature03461.html] S. Basu, Y. Gerchman, C. H. Collins, F. H. Arnold, in R. Weiss, „A synthetic multicellular system for programmed pattern formation“, Nature, Vol. 434, No. 7037, pp. 1130–1134, apr 2005.		[http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html] S. Basu, Y. Gerchman, C. H. Collins, F. H. Arnold, in R. Weiss, „A synthetic multicellular system for programmed pattern formation“, Nature, Vol. 434, No. 7037, pp. 1130–1134, apr 2005.

	[http://link.springer.com~~.nukweb.nuk.uni-lj.si~~/article/10.1007/s10126-001-0081-7] N.-S. Xia, W.-X. Luo, J. Zhang, X.-Y. Xie, H.-J. Yang, S.-W. Li, M. Chen, in M.-H. Ng, „Bioluminescence of Aequorea macrodactyla, a Common Jellyfish Species in the East China Sea“, Mar. Biotechnol., Vol. 4, No. 2, pp. 155–162, mar 2002.		[http://link.springer.com/article/10.1007/s10126-001-0081-7] N.-S. Xia, W.-X. Luo, J. Zhang, X.-Y. Xie, H.-J. Yang, S.-W. Li, M. Chen, in M.-H. Ng, „Bioluminescence of Aequorea macrodactyla, a Common Jellyfish Species in the East China Sea“, Mar. Biotechnol., Vol. 4, No. 2, pp. 155–162, mar 2002.