Device

Part:BBa_K228258

Designed by: Guosheng Zhang   Group: iGEM09_PKU_Beijing   (2009-09-20)

AND GATE AraC+SupD+Sal+RBS(B0033)+T7ptag

Part Main Page Protocol


This Device belongs to the SupD_T7ptag AND Gate family constructed by Peking University 2009 iGEM Team

PKU AND GATE FOR PARTS.png

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.

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_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 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 0.7045
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).

Safety

Because this part is from commonly used elements in bacteria. No safety problem can be arised by this part.



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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1274
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2144
    Illegal BamHI site found at 1214
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1976
    Illegal AgeI site found at 1320
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1323


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Categories
Parameters
n/aAND GATE AraC+SupD+Sal+RBS(B0033)+T7ptag