One of the Second AND Gate Design
Primarily we have 2 alternative design of the second AND Gate. One of the them employs the same mechanism as the first AND Gate. However, the activator is not T7 polymerase, it is a different activator with amber mutation of its Ser codon. Actually, we picked two candidates activator proteins. One of them is from partsregistry 2009 distribution, the PhiR73 delta activator and corresponding promoter PO promoter. The other is T3 polymerase and its promoter T3 promoter.
 fig1. One design of the second AND Gate
One of the input to the second AND Gate is from the ring signal. Because both of the AND Gates share the ring signal input, SupD tRNA is shared from AND Gate 1. The second AND Gate is intact only when coupled to the first AND Gate. The other input to the second AND Gate, of course, is from the memory module, we put a CI434 repressible and CI inducible promoter upstream of the PhiR73 delta/T3pol (amber mutation)protein.
When the bistable switch is in the “NO MEMORY” state, namely the CI434 state, PhiR73 delta/T3pol (amber mutation) mRNA is not expressed. At this time, ring signal alone, though expresses SupD tRNA, has no effect. When the bistable switch is turned to the “MEMORY” state, with the constitutive expression of CI and the increase of PhiR73 delta/T3pol mRNA level, the ring signal can lead to expression of functional PhiR73 delta/T3pol by rescuing the amber mutation in the PhiR73 delta/T3pol protein.
In order to creat a amber mutation inside of the PhiR73 delta/T3pol coding sequence, we carried out site directed mutagenesis with primers introduced with TAG amber mutation at the original Ser site. One problem that comes to us is how to decide which serine codon to mutate. In order to avoid that the half translated protein has function, we picked first few serine of the whole sequence of either PhiR73 delta activator and T3 polymerase. For PhiR73 delta it is on the first and second serine. (For PhiR73 delta sequence click [http://partsregistry.org/Part:BBa_I746352 Here]). In T3 polymerase, we picked four sites, they are on the 37th, 91th, 130th, 283th nucleotide of its coding sequence. The Sequence of the T3 polymerase with an amber mutation on its 37th and 91th nucleotide can be found in the parts that we hand in this year.
 fi2. Creating the Amber mutation
Another Design of the Second AND Gate
Another design of the second AND Gate is to make a hybrid promoter, which can be activated by salicylate, and repressed by CI434. This hybrid promoter regulates the expression of PhiR73 delta/T3pol (this time, no amber mutation).
Because what we take is the first strategy, the second design is just discussed briefly below:
First, we should figure out the -35 region and the -10 region of the salicylate inducible promoter. Then, the region between -35 and -10 is replaced with a CI434 binding site. Another CI434 binding site is placed downstream of -10 region. In this way, the promoter is silenced when CI434 is bound. After CI434 is removed from the system, this hybrid promoter still needs salicylate to activate.
 fig3. Another Design of the Second AND Gate
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