Part:BBa_K228260
AND GATE AraC+SupD+Sal+RBS(J44001)+T7ptag
This Device belongs to the SupD_T7ptag AND Gate family constructed by Peking University 2009 iGEM Team
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.
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 35.026100 39.460700 36.192900 14.883600 6.814620 10.218700 6.473800 0.1mM 30.315500 35.318700 20.343000 9.353110 4.865680 6.544900 4.752010 10μM 10.536600 12.382400 3.920270 2.500610 1.608520 1.465890 1.432810 1μM 4.745230 5.732030 3.760680 1.078570 0.870141 0.837732 0.709369 0.1μM 6.334980 6.302830 3.810920 0.943272 0.697559 0.942744 0.716153 10nM 5.166390 5.580230 2.713920 0.996142 0.883501 1.148130 0.806379 Control 6.979600 5.329700 3.870380 1.297480 0.835353 0.700015 0.895521
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
Table 3 Normalized fluorescence intensity for each inducer concentration combination GFP AraC 1mM 0.1mM 10μM 1μM 0.1μM 10nM Control Sal 1mM 38.575000 44.288103 41.938470 18.374815 7.614101 12.678288 7.153370 0.1mM 32.632400 39.773311 24.450721 10.677066 5.317683 7.412118 5.462080 10μM 11.465288 15.268064 4.689318 2.870964 2.062205 1.623355 1.559097 1μM 4.263459 5.903223 3.987996 1.161001 0.989922 0.937060 0.696826 0.1μM 6.696596 6.964453 4.330591 1.084221 0.780267 1.148287 0.883049 10nM 4.837444 5.636596 3.059662 1.210379 1.013189 1.284262 1.059631 Control 8.250118 6.147290 4.348742 1.540950 1.080664 0.786534 1.028153
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
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1274
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2144
Illegal BamHI site found at 1214 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1976
Illegal AgeI site found at 1320 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1323
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