Team:PKU Beijing/Parts Characterization/BBa K228260

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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.

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.

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_J44001 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 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		35.0261	39.4607	36.1929	14.8836	6.8146	10.2187	6.4738
0.1mM		30.3155	35.3187	20.3430	9.3531	4.8657	6.5449	4.7520
10μM		10.5366	12.3824	3.9203	2.5006	1.6085	1.4659	1.4328
1μM		4.7452	5.7320	3.7607	1.0786	0.8701	0.8377	0.7094
0.1μM		6.3350	6.3028	3.8109	0.9433	0.6976	0.9427	0.7162
10nM		5.1664	5.5802	2.7139	0.9961	0.8835	1.1481	0.8064
Control		6.9796	5.3297	3.8704	1.2975	0.8354	0.7000	0.8955

Table 2 Measured OD600 for each inducer concentration combination
OD600	AraC	1mM	0.1mM	10μM	1μM	0.1μM	10nM	Control
Sal 1mM		0.908	0.891	0.863	0.810	0.895	0.806	0.905
0.1mM		0.929	0.888	0.832	0.876	0.915	0.883	0.870
10μM		0.919	0.811	0.836	0.871	0.780	0.903	0.919
1μM		1.113	0.971	0.943	0.929	0.879	0.894	1.018
0.1μM		0.946	0.905	0.880	0.870	0.894	0.821	0.811
10nM		1.068	0.990	0.887	0.823	0.872	0.894	0.761
Control		0.846	0.867	0.890	0.842	0.773	0.890	0.871

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Table 3 Normalized fluorescence intensity for each inducer concentration combination
GFP	AraC 1mM	0.1mM	10μM	1μM	0.1μM	10nM	Control
Sal 1mM	38.575	44.288	41.938	18.375	7.614	12.678	7.153
0.1mM	32.632	39.773	24.451	10.677	5.318	7.412	5.462
10μM	11.465	15.268	4.689	2.871	2.062	1.623	1.559
1μM	4.263	5.903	3.988	1.161	0.990	0.937	0.697
0.1μM	6.697	6.964	4.331	1.084	0.780	1.148	0.883
10nM	4.837	5.637	3.060	1.210	1.013	1.284	1.060
Control	8.250	6.147	4.349	1.541	1.081	0.787	1.028

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 4. 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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 |[[Part:BBa_K228260|   Part Main Page  ]]||[[Part:BBa_K228260:Protocol|  Protocol   ]]
 |}
-{{PKU_Beijing/AND_GATE_FAMILY}}
 ==='''Description'''===
-We have adapted the original AND gate designed by Christopher A Voigt, which is published on Molecular Systems Biology, as the first module of our entire logic system. As the input module, 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, 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.
 [[Image:PKU_AND_Gate_Mechanism.png|600px|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''']]