Combinatorial synthesis of genetic networks - Revision history

MajaRemskar at 17:20, 25 January 2015

2015-01-25T17:20:56Z

← Older revision		Revision as of 17:20, 25 January 2015
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	[http://nemenmanlab.org/~ilya/images/9/99/Guet-etal-02.pdf Article] that I selected to explain was written by Guet C.C., Elowitz M.B., Hsing W. and Leibler S. and published in Science in 2002.		[http://nemenmanlab.org/~ilya/images/9/99/Guet-etal-02.pdf Article] that I selected to explain was written by Guet C.C., Elowitz M.B., Hsing W. and Leibler S. and published in Science in 2002.

			(Maja Remškar)

	== Introduction ==		== Introduction ==

MajaRemskar at 17:19, 25 January 2015

2015-01-25T17:19:40Z

← Older revision		Revision as of 17:19, 25 January 2015
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	[http://nemenmanlab.org/~ilya/images/9/99/Guet-etal-02.pdf Article] that I selected to explain was written by Guet C.C., Elowitz M.B., Hsing W. and Leibler S. and published in Science in 2002~~. It seems like it is quite important because until now it was cited 471 times~~.		[http://nemenmanlab.org/~ilya/images/9/99/Guet-etal-02.pdf Article] that I selected to explain was written by Guet C.C., Elowitz M.B., Hsing W. and Leibler S. and published in Science in 2002.

	== Introduction ==		== Introduction ==

MajaRemskar: /* Conclusion */

2015-01-04T18:35:35Z

Conclusion

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	=== Conclusion ===		=== Conclusion ===
	The present results show that really little of interacting genetic elements can generate a surprisingly large diversity of complex behaviors. The current system uses a small number of building blocks restricted only to transcriptional regulation. Both the number of elements and the range of biochemical interactions can be extended by including other modular genetic elements [3].		The present results show that really little of interacting genetic elements can generate a surprisingly large diversity of complex behaviors. The current system uses a small number of building blocks restricted only to transcriptional regulation. Both the number of elements and the range of biochemical interactions can be extended by including other modular genetic elements [3].

	There are also some ideas for the future. The approach can be taken beyond the intracellular level by linking input and output through cell-cell signaling molecules, such as those involved in quorum sensing [3]. The latter is a system of stimulus and response correlated to population density. Many species of bacteria use quorum sensing to coordinate gene expression according to the density of their local population [28]. Lastly, this combinatorial strategy can be used to search for other dynamic behaviors such as switches, sensors, oscillators, and amplifiers, as well as for high-level structural properties, such as robustness or noise-resistance [29].		There are also some ideas for the future. The approach can be taken beyond the intracellular level by linking input and output through cell-cell signaling molecules, such as those involved in quorum sensing [3]. The latter is a system of stimulus and response correlated to population density. Many species of bacteria use quorum sensing to coordinate gene expression according to the density of their local population [28]. Lastly, this combinatorial strategy can be used to search for other dynamic behaviors such as switches, sensors, oscillators, and amplifiers, as well as for high-level structural properties, such as robustness or noise-resistance [29].

MajaRemskar: /* Transcriptional regulators */

2015-01-04T17:42:26Z

Transcriptional regulators

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	Transcription is the first step of gene expression, in which a particular segment of DNA is copied into RNA by the enzyme RNA polymerase. Regulation of transcription is highly controlled process and there are plenty of different molecules involved. Transcriptional regulators control the rate of gene transcription for example by helping or hindering RNA polymerase binding to DNA. A single gene can be regulated in a range of ways, from altering the number of copies of RNA that are transcribed, to the temporal control of when the gene is transcribed. This control allows the cell or organism to respond to a variety of intra- and extracellular signals and thus respond adequately. For example, mRNA is produced to encode enzymes to adapt to a change in a food source, producing the gene products involved in cell cycle specific activities, and producing the gene products responsible for cellular differentiation in higher eukaryotes [4].		Transcription is the first step of gene expression, in which a particular segment of DNA is copied into RNA by the enzyme RNA polymerase. Regulation of transcription is highly controlled process and there are plenty of different molecules involved. Transcriptional regulators control the rate of gene transcription for example by helping or hindering RNA polymerase binding to DNA. A single gene can be regulated in a range of ways, from altering the number of copies of RNA that are transcribed, to the temporal control of when the gene is transcribed. This control allows the cell or organism to respond to a variety of intra- and extracellular signals and thus respond adequately. For example, mRNA is produced to encode enzymes to adapt to a change in a food source, producing the gene products involved in cell cycle specific activities, and producing the gene products responsible for cellular differentiation in higher eukaryotes [4].

	== Transcriptional regulators ==		=== Transcriptional regulators ===
	Transcriptional regulators are transcription factors (TF) and other proteins working in concert to finely tune the amount of RNA being produced through a variety of mechanisms [4]. The fact is that any given gene is likely controlled by a specific combination of factors, which is called combinatorial control. In a hypothetical example, the factors A and B might regulate a distinct set of genes from the combination of factors A and C. This combinatorial nature extends to complexes of far more than two proteins, and allows a very small subset (less than 10%) of the genome to control the transcriptional program of the entire cell [4]. All three of TFs used in this experiment are shortly described below.		Transcriptional regulators are transcription factors (TF) and other proteins working in concert to finely tune the amount of RNA being produced through a variety of mechanisms [4]. The fact is that any given gene is likely controlled by a specific combination of factors, which is called combinatorial control. In a hypothetical example, the factors A and B might regulate a distinct set of genes from the combination of factors A and C. This combinatorial nature extends to complexes of far more than two proteins, and allows a very small subset (less than 10%) of the genome to control the transcriptional program of the entire cell [4]. All three of TFs used in this experiment are shortly described below.

MajaRemskar: /* References */

2015-01-04T17:41:28Z

References

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	<span style="font-size:94%">29. Hartwell, L.H., et al, From molecular to modular cell biology. Nature, 1999, vol. 402(6761 Suppl), p. C47-52.</span>		<span style="font-size:94%">29. Hartwell, L.H., et al, From molecular to modular cell biology. Nature, 1999, vol. 402(6761 Suppl), p. C47-52.</span>


	[[SB students resources]]		[[SB students resources]]

MajaRemskar: /* Methods */

2015-01-04T17:40:37Z

Methods

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	In Figure 1, they present modular genetic cloning strategy used to generate combinatorial libraries of logical circuits. At first (A) all 15 possible promoter-gene units were built. Individual promoters and genes were than amplified by PCR. The genes [denoted ''-lite'' in (B)] have an ssrA tag that reduces the half-life of the proteins encoded by the modified gene [12]. The five promoters used were P<span style="font-size:80%">L1</span> and P<span style="font-size:80%">L2</span> (repressed by LacI), P<span style="font-size:80%">T</span> (repressed by TetR), and P<span style="font-size:80%">λ-</span> and P<span style="font-size:80%">λ+</span> (repressed and activated by λ cI). The transcriptional terminator T1 was present at the end of each gene [3].		In Figure 1, they present modular genetic cloning strategy used to generate combinatorial libraries of logical circuits. At first (A) all 15 possible promoter-gene units were built. Individual promoters and genes were than amplified by PCR. The genes [denoted ''-lite'' in (B)] have an ssrA tag that reduces the half-life of the proteins encoded by the modified gene [12]. The five promoters used were P<span style="font-size:80%">L1</span> and P<span style="font-size:80%">L2</span> (repressed by LacI), P<span style="font-size:80%">T</span> (repressed by TetR), and P<span style="font-size:80%">λ-</span> and P<span style="font-size:80%">λ+</span> (repressed and activated by λ cI). The transcriptional terminator T1 was present at the end of each gene [3].

	They amplified promotor region and separately the region with the gene. Identical RBS sites were used as internal primers for the subsequent fusion PCR step to form promoter-gene units [13]. In order to control the number of promoter-gene units and the position of a given gene in the network, Bgl I sites were incorporated in PCR primers, as shown. The special recognition and restriction properties of Bgl I [14] allow various sticky ends to be produced by Bgl I cleavage. Here, they designed the Bgl I sites such that specific cohesive ends x and y were associated with each regulatory gene (for lacI, x<span style="font-size:80%">lac</span> = GCC, y<span style="font-size:80%">lac</span> = TTC; for λ cI, x<span style="font-size:80%">cI</span> = AAG, y<span style="font-size:80%">cI</span> = GTG; and for tetR, x<span style="font-size:80%">tet</span> = CAC, y<span style="font-size:80%">tet</span> = TCG). You can notice that y<span style="font-size:80%">lac</span>lac is compatible with x<span style="font-size:80%">cI</span>, y<span style="font-size:80%">cI</span> is compatible with x<span style="font-size:80%">~~tetR~~</span>, and so on [3].		They amplified promotor region and separately the region with the gene. Identical RBS sites were used as internal primers for the subsequent fusion PCR step to form promoter-gene units [13]. In order to control the number of promoter-gene units and the position of a given gene in the network, Bgl I sites were incorporated in PCR primers, as shown. The special recognition and restriction properties of Bgl I [14] allow various sticky ends to be produced by Bgl I cleavage. Here, they designed the Bgl I sites such that specific cohesive ends x and y were associated with each regulatory gene (for lacI, x<span style="font-size:80%">lac</span> = GCC, y<span style="font-size:80%">lac</span> = TTC; for λ cI, x<span style="font-size:80%">cI</span> = AAG, y<span style="font-size:80%">cI</span> = GTG; and for tetR, x<span style="font-size:80%">tet</span> = CAC, y<span style="font-size:80%">tet</span> = TCG). You can notice that y<span style="font-size:80%">lac</span>lac is compatible with x<span style="font-size:80%">cI</span>, y<span style="font-size:80%">cI</span> is compatible with x<span style="font-size:80%">tet</span>, and so on [3].

	In step B when all 15 possible fusion PCR products were mixed together and ligated, the resulting products contained exactly three promoter-gene units in one particular order (lacI, λ cI, tetR). These products were cloned into a low copy number plasmid (3-4 copies/cell) [15], carrying the reporter gene ''gfpmut3'' under the control of Pλ- , which is a fourth transcriptional unit coding for green fluorescent protein (GFP) controlled by the λ cI repressible promoter. The fluorescent signal acts as the network output, whereas the levels of the two chemical inducers were used as inputs [3].		In step B when all 15 possible fusion PCR products were mixed together and ligated, the resulting products contained exactly three promoter-gene units in one particular order (lacI, λ cI, tetR). These products were cloned into a low copy number plasmid (3-4 copies/cell) [15], carrying the reporter gene ''gfpmut3'' under the control of Pλ- , which is a fourth transcriptional unit coding for green fluorescent protein (GFP) controlled by the λ cI repressible promoter. The fluorescent signal acts as the network output, whereas the levels of the two chemical inducers were used as inputs [3].

MajaRemskar: /* Methods */

2015-01-04T17:40:19Z

Methods

← Older revision		Revision as of 17:40, 4 January 2015
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	In Figure 1, they present modular genetic cloning strategy used to generate combinatorial libraries of logical circuits. At first (A) all 15 possible promoter-gene units were built. Individual promoters and genes were than amplified by PCR. The genes [denoted ''-lite'' in (B)] have an ssrA tag that reduces the half-life of the proteins encoded by the modified gene [12]. The five promoters used were P<span style="font-size:80%">L1</span> and P<span style="font-size:80%">L2</span> (repressed by LacI), P<span style="font-size:80%">T</span> (repressed by TetR), and P<span style="font-size:80%">λ-</span> and P<span style="font-size:80%">λ+</span> (repressed and activated by λ cI). The transcriptional terminator T1 was present at the end of each gene [3].		In Figure 1, they present modular genetic cloning strategy used to generate combinatorial libraries of logical circuits. At first (A) all 15 possible promoter-gene units were built. Individual promoters and genes were than amplified by PCR. The genes [denoted ''-lite'' in (B)] have an ssrA tag that reduces the half-life of the proteins encoded by the modified gene [12]. The five promoters used were P<span style="font-size:80%">L1</span> and P<span style="font-size:80%">L2</span> (repressed by LacI), P<span style="font-size:80%">T</span> (repressed by TetR), and P<span style="font-size:80%">λ-</span> and P<span style="font-size:80%">λ+</span> (repressed and activated by λ cI). The transcriptional terminator T1 was present at the end of each gene [3].

	They amplified promotor region and separately the region with the gene. Identical RBS sites were used as internal primers for the subsequent fusion PCR step to form promoter-gene units [13]. In order to control the number of promoter-gene units and the position of a given gene in the network, Bgl I sites were incorporated in PCR primers, as shown. The special recognition and restriction properties of Bgl I [14] allow various sticky ends to be produced by Bgl I cleavage. Here, they designed the Bgl I sites such that specific cohesive ends x and y were associated with each regulatory gene (for lacI, ~~xlac~~ = GCC, ~~ylac~~ = TTC; for λ cI, ~~xcI~~ = AAG, ~~ycI~~ = GTG; and for tetR, ~~xtet~~ = CAC, ~~ytet~~ = TCG). You can notice that ~~ylac~~ is compatible with ~~xcI~~, ~~ycI~~ is compatible with ~~xtetR~~, and so on [3].		They amplified promotor region and separately the region with the gene. Identical RBS sites were used as internal primers for the subsequent fusion PCR step to form promoter-gene units [13]. In order to control the number of promoter-gene units and the position of a given gene in the network, Bgl I sites were incorporated in PCR primers, as shown. The special recognition and restriction properties of Bgl I [14] allow various sticky ends to be produced by Bgl I cleavage. Here, they designed the Bgl I sites such that specific cohesive ends x and y were associated with each regulatory gene (for lacI, x<span style="font-size:80%">lac</span> = GCC, y<span style="font-size:80%">lac</span> = TTC; for λ cI, x<span style="font-size:80%">cI</span> = AAG, y<span style="font-size:80%">cI</span> = GTG; and for tetR, x<span style="font-size:80%">tet</span> = CAC, y<span style="font-size:80%">tet</span> = TCG). You can notice that y<span style="font-size:80%">lac</span>lac is compatible with x<span style="font-size:80%">cI</span>, y<span style="font-size:80%">cI</span> is compatible with x<span style="font-size:80%">tetR</span>, and so on [3].

	In step B when all 15 possible fusion PCR products were mixed together and ligated, the resulting products contained exactly three promoter-gene units in one particular order (lacI, λ cI, tetR). These products were cloned into a low copy number plasmid (3-4 copies/cell) [15], carrying the reporter gene ''gfpmut3'' under the control of Pλ- , which is a fourth transcriptional unit coding for green fluorescent protein (GFP) controlled by the λ cI repressible promoter. The fluorescent signal acts as the network output, whereas the levels of the two chemical inducers were used as inputs [3].		In step B when all 15 possible fusion PCR products were mixed together and ligated, the resulting products contained exactly three promoter-gene units in one particular order (lacI, λ cI, tetR). These products were cloned into a low copy number plasmid (3-4 copies/cell) [15], carrying the reporter gene ''gfpmut3'' under the control of Pλ- , which is a fourth transcriptional unit coding for green fluorescent protein (GFP) controlled by the λ cI repressible promoter. The fluorescent signal acts as the network output, whereas the levels of the two chemical inducers were used as inputs [3].

MajaRemskar: /* Methods */

2015-01-04T17:38:30Z

Methods

← Older revision		Revision as of 17:38, 4 January 2015
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	=== Methods ===		=== Methods ===
	In Figure 1, they present modular genetic cloning strategy used to generate combinatorial libraries of logical circuits. At first (A) all 15 possible promoter-gene units were built. Individual promoters and genes were than amplified by PCR. The genes [denoted ''-lite'' in (B)] have an ssrA tag that reduces the half-life of the proteins encoded by the modified gene [12]. The five promoters used were ~~PL1~~ and ~~PL2~~ (repressed by LacI), PT (repressed by TetR), and Pλ - and Pλ+ (repressed and activated by λ cI). The transcriptional terminator T1 was present at the end of each gene [3].		In Figure 1, they present modular genetic cloning strategy used to generate combinatorial libraries of logical circuits. At first (A) all 15 possible promoter-gene units were built. Individual promoters and genes were than amplified by PCR. The genes [denoted ''-lite'' in (B)] have an ssrA tag that reduces the half-life of the proteins encoded by the modified gene [12]. The five promoters used were P<span style="font-size:80%">L1</span> and P<span style="font-size:80%">L2</span> (repressed by LacI), P<span style="font-size:80%">T</span> (repressed by TetR), and P<span style="font-size:80%">λ-</span> and P<span style="font-size:80%">λ+</span> (repressed and activated by λ cI). The transcriptional terminator T1 was present at the end of each gene [3].

	They amplified promotor region and separately the region with the gene. Identical RBS sites were used as internal primers for the subsequent fusion PCR step to form promoter-gene units [13]. In order to control the number of promoter-gene units and the position of a given gene in the network, Bgl I sites were incorporated in PCR primers, as shown. The special recognition and restriction properties of Bgl I [14] allow various sticky ends to be produced by Bgl I cleavage. Here, they designed the Bgl I sites such that specific cohesive ends x and y were associated with each regulatory gene (for lacI, xlac = GCC, ylac = TTC; for λ cI, xcI = AAG, ycI = GTG; and for tetR, xtet = CAC, ytet = TCG). You can notice that ylac is compatible with xcI, ycI is compatible with xtetR, and so on [3].		They amplified promotor region and separately the region with the gene. Identical RBS sites were used as internal primers for the subsequent fusion PCR step to form promoter-gene units [13]. In order to control the number of promoter-gene units and the position of a given gene in the network, Bgl I sites were incorporated in PCR primers, as shown. The special recognition and restriction properties of Bgl I [14] allow various sticky ends to be produced by Bgl I cleavage. Here, they designed the Bgl I sites such that specific cohesive ends x and y were associated with each regulatory gene (for lacI, xlac = GCC, ylac = TTC; for λ cI, xcI = AAG, ycI = GTG; and for tetR, xtet = CAC, ytet = TCG). You can notice that ylac is compatible with xcI, ycI is compatible with xtetR, and so on [3].

MajaRemskar: /* Discussion */

2015-01-04T17:34:35Z

Discussion

← Older revision		Revision as of 17:34, 4 January 2015
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	Combinatorial techniques inspired by recombination, such as DNA shuffling, have often proven successful in enhancing or changing the enzymatic activities of proteins and pathways [24, 25] without requiring an understanding of the mechanisms by which they work. DNA shuffling is a way to rapidly propagate beneficial mutations in a directed evolution experiment [26]. It is used to rapidly increase DNA library size [27] because it is a recombination between different DNA species with different mutations [26]. However, combinatorial methods in simple and well-controlled systems can and should also be used to gain better understanding of system and level properties of cellular networks for further practical applications [3].		Combinatorial techniques inspired by recombination, such as DNA shuffling, have often proven successful in enhancing or changing the enzymatic activities of proteins and pathways [24, 25] without requiring an understanding of the mechanisms by which they work. DNA shuffling is a way to rapidly propagate beneficial mutations in a directed evolution experiment [26]. It is used to rapidly increase DNA library size [27] because it is a recombination between different DNA species with different mutations [26]. However, combinatorial methods in simple and well-controlled systems can and should also be used to gain better understanding of system and level properties of cellular networks for further practical applications [3].

			=== Conclusion ===
	The present results show that really little of interacting genetic elements can generate a surprisingly large diversity of complex behaviors. The current system uses a small number of building blocks restricted only to transcriptional regulation. Both the number of elements and the range of biochemical interactions can be extended by including other modular genetic elements [3].		The present results show that really little of interacting genetic elements can generate a surprisingly large diversity of complex behaviors. The current system uses a small number of building blocks restricted only to transcriptional regulation. Both the number of elements and the range of biochemical interactions can be extended by including other modular genetic elements [3].
	There are also some ideas for the future. The approach can be taken beyond the intracellular level by linking input and output through cell-cell signaling molecules, such as those involved in quorum sensing [3]. The latter is a system of stimulus and response correlated to population density. Many species of bacteria use quorum sensing to coordinate gene expression according to the density of their local population [28]. Lastly, this combinatorial strategy can be used to search for other dynamic behaviors such as switches, sensors, oscillators, and amplifiers, as well as for high-level structural properties, such as robustness or noise-resistance [29].		There are also some ideas for the future. The approach can be taken beyond the intracellular level by linking input and output through cell-cell signaling molecules, such as those involved in quorum sensing [3]. The latter is a system of stimulus and response correlated to population density. Many species of bacteria use quorum sensing to coordinate gene expression according to the density of their local population [28]. Lastly, this combinatorial strategy can be used to search for other dynamic behaviors such as switches, sensors, oscillators, and amplifiers, as well as for high-level structural properties, such as robustness or noise-resistance [29].

MajaRemskar: /* References */

2015-01-04T17:31:04Z

References

← Older revision		Revision as of 17:31, 4 January 2015
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	<span style="font-size:94%">29. Hartwell, L.H., et al, From molecular to modular cell biology. Nature, 1999, vol. 402(6761 Suppl), p. C47-52.</span>		<span style="font-size:94%">29. Hartwell, L.H., et al, From molecular to modular cell biology. Nature, 1999, vol. 402(6761 Suppl), p. C47-52.</span>


			[[SB students resources]]