Team:PKU Beijing/Project/AND Gate 1/Design

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Thus the T7ptag and supD gene are made into standard parts. Because our project employs many parts and devices, it is important for us to prevent these parts to interfere with each other. So the First thing after the core parts are constructed, we put a strong terminator BBa_B0015 downstream of the two parts.
Thus the T7ptag and supD gene are made into standard parts. Because our project employs many parts and devices, it is important for us to prevent these parts to interfere with each other. So the First thing after the core parts are constructed, we put a strong terminator BBa_B0015 downstream of the two parts.
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[[Image:T7ptag_terminater_construction.png|600px|left|thumb|fig9. Add Terminator downstream of T7ptag]]
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[[Image:T7ptag_terminater_construction.png|400px|left|thumb|fig9. Add Terminator downstream of T7ptag]]
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[[Image:SupD_Terminater_Construction.png|400px|left|thumb|fig10. Add Terminator downstream of T7ptag]]
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Revision as of 16:41, 10 October 2009

 
Project > AND Gate 1 > Design

Mechanism

Many AND Gate have been designed and constructed in the past years. In our project, We adopted the design of Christopher Voigt, Which is published on Molecular Systems Biology.

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

Fig.1 illustrates the mechanism of the design. One of the inputs of the and gate leads to the expression of T7ptag mRNA which is a T7 polymerase coding sequence with amber mutations inside. The other input of the And gate leads to the expression of supD tRNA, which is a amber mutation suppressor. The concurrence of the mRNA and the tRNA can make functional T7 polymerase, which can activate the downstream T7 promoter.

The AND Gate can be further decoupled into a input interface, a core, and a output interface.

fig2. AND Gate Biological Components

The input interface includes two inducible promoter which can be induced to expression by two different environmental small molecules. Core of the AND Gate is the T7 polymerase coding sequence with amber mutations and supD tRNA. And the Output interface is actually a T7 promoter, which regulate the expression of the downstream component.

Sensor System (The input interface)

The input interface includes two inducible promoter which can be induced to expression by two different environmental small molecules.

We actually prepared five inducible promoter, and make combinations among the five promoters. The five promoters are tetR repressible promoter inducible by aTc, PBad promoter by arabinose, Lac Promoter inducible by IPTG, Salicylate inducible promoter and Lux promoter that responses to HomoSerine Lactone.

All of these inducible promoters can only work in the presence of activators or repressors, so we cloned all the activators and the repressors downstream of a constitutive promoter to make a protein generator. Then the generator is placed upstream of the corresponding inducible promoter.

There are two ways, actually, to put the generator upstream of the inducible promoter. As shown in fig3.

fig3A. One Construct
fig3B. Another Construct

1. The constitutive promoter is cloned back to back with the inducible promoter, thus transcription of the activator or the repressor is opposite of the inducible promoter. 2. The generator transcripts in the same direction of the inducible promoter, the constitutive promoter is prevented to interfere with the inducible promoter by the terminator. However, any terminator is leaky, the first way is the better way to prevent interference.

This construct combines repressors (activators) together with its promoter. It is actually a quite useful part even outside of the AND gate Module. We call it an environmental small molecule sensor. It provides a sensor for environmental signals and a interface to intracellular circuits. Actually, a similar construct(repressor protein only) without the constitutive promoter is called an inverter, but we think it is more proper to be called a sensor when the promoter upstream of the repressor is a constitutive promoter. Its signaling molecule like aTc, IPTG, arabinose, HomoSerineLactone and salicylate diffuse into the cell and take function. It is like the some of the hydrophobic hormones in high organisms.

The promoters we chose at first is the LuxR-LuxP construct induced by Homoserinelactone, the tet operon induced by aTc, the Lac operon induced by IPTG, and the Arabinose inducible promoter PBad. So we designed the following sensors:

fig4. aTc Sensor




The aTc Sensor is a tet promoter repressible by tetR protein. The repression is released in the presence of aTc.




fig5. IPTG Sensor




The IPTG Sensor is a lac promoter repressible by lacI repressor.





fig6. HSL Sensor



And because the LuxR-LuxP sensor has already been constructed by iGEM2006_PennState. So we didn’t reverse the LuxR coding sequence but just use their construct.




The salicylate Sensor: The salicylate Sensor is not made up of standard parts, it is originally on the pSal-SupD plasmid that Voigt Lab mailed us. We got the sequence from NCIBI there is no standard enzymes inside of its sequence. We designed primers and PCR it from the plasmid. Then the promter is placed upstream of part E0840 to characterize its induction curve.

Core of the AND Gate

SupD tRNA and T7ptag mRNA makes the core of this AND Gate. T7ptag gene is a T7 polymerase coding gene with two amber mutation at it Serine codon. The mutated T7 polymerase gene can only be transcribed but cannot be successfully expressed for the failure of translation. The SupD tRNA is an amber mutation suppressor, this tRNA recognizes the TAG stop codon and mandatorily translate it into a Serine. Thus with supD, the function of the T7 polymerase gene is recovered. According to the Paper "Environmental signal integration by a modular AND Gate", SupD tRNA has little side effect on E.coli, although it can suppress normal termination of translation.

We Use PCR to make T7ptag and SupD into standard biobrick. The primer is designed according to the paper, we keep the complementary sequence in the original primer, and replace the enzyme cut site in the original primer with standard prefix and suffix. The original plasmid where we get SupD sequence and T7ptag sequence is shown below:

fig8. The Original Plasmid Where We Got the SupD and T7ptag Gene

Thus the T7ptag and supD gene are made into standard parts. Because our project employs many parts and devices, it is important for us to prevent these parts to interfere with each other. So the First thing after the core parts are constructed, we put a strong terminator BBa_B0015 downstream of the two parts.

fig9. Add Terminator downstream of T7ptag
fig10. Add Terminator downstream of T7ptag




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