Difference between revisions of "Part:BBa K228258"

Revision as of 12:57, 20 October 2009

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

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

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)

|+Table 1. Measured fluorescence intensity for each inducer concentration combination

Fluorescence intensity	AraC 1mM	0.1mM	10μM	1μM	0.1μM	10nM	Control
Sal 1mM	16.184800	17.902000	14.127100	3.846290	1.894130	1.503850	1.317190
0.1mM	12.610800	9.890710	8.623620	2.018330	1.118300	1.187150	1.214980
10μM	3.693290	3.783040	2.079610	0.883882	0.881562	0.719105	0.742025
1μM	1.346850	1.321000	1.020660	0.704721	0.943905	0.795452	0.563781
0.1μM	1.187240	1.108090	1.163110	0.993399	0.655787	0.600689	0.728444
10nM	0.934812	1.102860	0.883133	0.901533	0.948425	0.532484	0.704519
Control	1.258520	1.523960	1.135110	0.876301	0.851688	0.541163	0.722297

Fig 1. GFP expression of AND gate, K228258: 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, K228258: 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

Sequence and Features