Team:PKU Beijing/Parts Characterization/BBa K228258

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Description

This AND Gate is designed by Christopher A Voigt(Molecular Systems Biology 3:133, 2007). We reconstructed the AND gate according to the principle of standard assembly, and finally greatly improved its efficiency. In brief, the AND gate is "open" only when both inputs are present. The AND gate accepts small molecules as inputs and integrate the signals to activate T7 promoter, so as to trigger the expression of downstream gene.

Fig1. The mechanism of the AND Gate J Christopher Anderson, Christopher A Voigt and Adam P Arkin (2007) Environmental signal integration by a modular AND gate Molecular Systems Biology 3:133

The original design of the synthetic AND gate uses two promoters as input sensors and also a promoter as output. The AND gate function is obtained via an interaction between a nonsense mutated DNA and a mutation suppressor tRNA. The ﬁrst promoter drives the transcription of the T7 RNA polymerase sequence which includes two internal amber codons (T7ptag) at positions 8 and 14 respectively, which is mutated from a normal codon due to base substitution. In wild-type E.coli ,the amber codon (TAG) would be decoded and thus stop the translation, producing nonfunctional polypeptide, and the double mutations can offer the lowest background level of normal T7 RNA polymerase, which would make the next step of regulation of the AND gate more convenient. And the second promoter regulates an amber suppressor gene supD, which produces a specific tRNA decoding TAG as serine. Therefore, only when both of the inputs are given, the normal T7 RNA polymerase can be synthesized, then activates the T7 promoter and express the downstream gene.

In order to make the AND gate inducible, the researchers chose two promoters that can be induced by specific small molecules. And inspired by the conception of modulation put forward by the authors, we have swapped the promoters, expecting to obtain a better AND gate. Because the core interactions between the amber mutated DNA and the suppressor tRNA are not influenced, the alternation of promoters would not change the basic AND logic behavior. In addition, we also swapped various RBSs of T7ptag to regulate the relative quantity of mutated T7 polymerase and supD tRNA and thus facilitated the screening of a better AND gate.

To see more details...

This gate is one of the best AND gates we obtained, of which T7ptagn gene is regulated by BBa_B0033 RBS.

In order to characterize the AND gate quantificationally, we placed GFP coding gene downstream of the T7 promoter and measured the output fluorescence intensity. The input promoters are activated in different degrees by varying the concentration of the arabinose and salicylate inducers from 10^-7 to 10^-3. (For more details, refer to our protocol)

Result

Table 1. Measured fluorescence intensity for each inducer concentration combination
GFP	AraC	1mM	0.1mM	10μM	1μM	0.1μM	10nM	Control
Sal 1mM		16.1848	17.9020	14.1271	3.8463	1.8941	1.5039	1.3172
0.1mM		12.6108	9.8907	8.6236	2.0183	1.1183	1.1872	1.2150
10μM		3.6933	3.7830	2.0796	0.8839	0.8816	0.7191	0.7420
1μM		1.3469	1.3210	1.0207	0.7047	0.9439	0.7955	0.5638
0.1μM		1.1872	1.1081	1.1631	0.9934	0.6558	0.6007	0.7284
10nM		0.9348	1.1029	0.8831	0.9015	0.9484	0.5325
Control		1.2585	1.5240	1.1351	0.8763	0.8517	0.5412	0.7223

Table 2 Measured OD600 for each inducer concentration combination
OD600	AraC	1mM	0.1mM	10μM	1μM	0.1μM	10nM	Control
Sal 1mM		0.799	0.727	0.765	0.736	0.748	0.755	0.761
0.1mM		0.879	0.757	0.755	0.775	0.759	0.761	0.775
10μM		0.883	0.760	0.745	0.776	0.780	0.710	0.731
1μM		1.087	0.794	0.787	0.781	0.775	0.766	0.746
0.1μM		0.888	0.763	0.756	0.751	0.770	0.740	0.773
10nM		0.909	0.744	0.732	0.751	0.693	0.721	0.770
Control		0.886	0.773	0.767	0.689	0.759	0.710	0.747

Table 3 Normalized fluorescence intensity for each inducer concentration combination
GFP	AraC 1mM	0.1mM	10μM	1μM	0.1μM	10nM	Control
Sal 1mM	20.256	24.624	18.467	5.226	2.532	1.992	1.731
0.1mM	14.347	13.066	11.422	2.604	1.473	1.560	1.568
10μM	4.183	4.978	2.791	1.139	1.130	1.013	1.015
1μM	1.239	1.664	1.297	0.902	1.218	1.038	0.756
0.1μM	1.337	1.452	1.539	1.323	0.852	0.812	0.942
10nM	1.028	1.482	1.206	1.200	1.369	0.739	0.915
Control	1.420	1.971	1.480	1.272	1.122	0.762	0.967

Fig 2. GFP expression of AND gate, K228260: AraC-SupD-Sal-T7ptag (rbs: J44001). The data are showed for (bottom to top): 1×10^-8M, 1×10^-7M, 1×10^-6M, 1×10^-5M, 1×10^-4M and 1×10^-3M salicylate, and (left to right) 1×10^-8M, 1×10^-7M, 1×10^-6M, 1×10^-5M, 1×10^-4M and 1×10^-3M arabinose. The Z axis denotes the value of fluorescence normalized by the OD600 value.

Fig 3. GFP expression of AND gate, K228260: simplified version of Fig2. The data are showed for 1×10^-8 and 1×10^-3M salicylate, and 1×10^-7 and 1×10^-3M arabinose. The concentration of 1×10^-7M is regarded as input=0, and 1×10^-3M is regarded as input=1. So the four columns show the response of (0,0), (0,1), (1,0) and (1,1).

For more information: refer to K228000(T7ptag) and K228001(SupD) referrence: Anderson JC, Voigt CA, Arkin AP (2007) Environmental signal integration by a modular AND gate. Mol Syst Biol 3: 133

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@@ Line 13: / Line 13: @@
 ==='''Description'''===
-This AND Gate is designed by Christopher A Voigt, which is published on Molecular Systems Biology. We standarlized and modified the input, and finally greatly improved the AND Gate. The AND gate accepts the small molecules as inputs and integrate the signals to activate the T7 promoter, thus triggering the expression of downstream gene.
+This AND Gate is designed by Christopher A Voigt(Molecular Systems Biology 3:133, 2007). We reconstructed the AND gate according to the principle of standard assembly, and finally greatly improved its efficiency. In brief, the AND gate is "open" only when both inputs are present. The AND gate accepts small molecules as inputs and integrate the signals to activate T7 promoter, so as to trigger the expression of downstream gene.
 [[Image:PKU_AND_Gate_Mechanism.png|400px|center|thumb|Fig1. '''The mechanism of the AND Gate''' J Christopher Anderson, Christopher A Voigt and Adam P Arkin (2007) ''Environmental signal integration by a modular AND gate'' '''Molecular Systems Biology 3:133''']]
-The original design of the synthetic AND gate uses two promoters as input sensors and also a promoter as output. The AND gate function is obtained via an interaction between a nonsense mutated DNA and a mutation suppressor tRNA. The ﬁrst promoter drives the transcription of the T7 RNA polymerase gene with two internal amber codons (T7ptag) at positions 8 and 14 respectively, which is mutated from a normal codon due to base substitution. In wild-type E.coli ,the amber codon (TAG) would be decoded and thus stop the translation, producing nonfunctional polypeptide, and the double mutations can offer the lowest background level of normal T7 RNA polymerase, which would make the next regulation of the AND gate more convenient. And the second promoter regulates an amber suppressor gene supD, which produces a specific tRNA decoding TAG as serine. Therefore, only when both of the inputs are given, the normal T7 RNA polymerase can be synthesized, then activates the T7 promoter and express the downstream gene.
+The original design of the synthetic AND gate uses two promoters as input sensors and also a promoter as output. The AND gate function is obtained via an interaction between a nonsense mutated DNA and a mutation suppressor tRNA. The ﬁrst promoter drives the transcription of the T7 RNA polymerase sequence which includes two internal amber codons (T7ptag) at positions 8 and 14 respectively, which is mutated from a normal codon due to base substitution. In wild-type E.coli ,the amber codon (TAG) would be decoded and thus stop the translation, producing nonfunctional polypeptide, and the double mutations can offer the lowest background level of normal T7 RNA polymerase, which would make the next step of regulation of the AND gate more convenient. And the second promoter regulates an amber suppressor gene supD, which produces a specific tRNA decoding TAG as serine. Therefore, only when both of the inputs are given, the normal T7 RNA polymerase can be synthesized, then activates the T7 promoter and express the downstream gene.
 In order to make the AND gate inducible, the researchers chose two promoters that can be induced by specific small molecules. And inspired by the conception of modulation put forward by the authors, we have swapped the promoters, expecting to obtain a better AND gate. Because the core interactions between the amber mutated DNA and the suppressor tRNA are not influenced, the alternation of promoters would not change the basic AND logic behavior. In addition, we also swapped various RBSs of T7ptag to regulate the relative quantity of mutated T7 polymerase and supD tRNA and thus facilitated the screening of a better AND gate.
@@ Line 25: / Line 25: @@
 This gate is one of the best AND gates we obtained, of which T7ptagn gene is regulated by BBa_B0033 RBS.
-In order to characterize the AND gate quantificationally, we cloned a reporter gene gfp downstream of the T7 promoter to measure the output fluorescence intensity. The input promoters are activated in different degrees by varying the concentration of the arabinose and salicylate inducers from 10^-7 to 10^-3. (For more detail, refer to our [[Team:PKU_Beijing/Parts_Characterization/BBa_K228258/Protocol|protocol]])
+In order to characterize the AND gate quantificationally, we placed GFP coding gene downstream of the T7 promoter and measured the output fluorescence intensity. The input promoters are activated in different degrees by varying the concentration of the arabinose and salicylate inducers from 10^-7 to 10^-3. (For more details, refer to our [[Team:PKU_Beijing/Parts_Characterization/BBa_K228258/Protocol|protocol]])
 ==='''Result'''===