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Revision as of 16:28, 29 October 2006

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AND gate

The AND gate's PoPs output activity should be correlated to the PoPs input activity as shown in the picture:

 input A ^
         | L  D  H
         | D  D  D
         | L  D  L
         +--------->
            input B

 output: High, Low, Dont care

Implementation alternatives

Part to be used in the prototype system: [http://partsregistry.org/Part:BBa_J34100 BBa_J34100]

An AND gate produces an output only if both input signals are present. We want to implement such a “device” by using a suppressor tRNA. In our proposed model Signal 1 would lead to the transcription of the T7 RNA polymerase. However, an early stop codon was introduced into the coding sequence, leading to the expression of a truncated, non-functional protein. Signal 2, on the other hand, will lead to the expression of a suppressor tRNA. This tRNA recognizes the stop codon, preventing the end of translation and leading to incorporation of a glutamine into the nascent amino acid chain, so that translation can be completed. Consequently, a functional T7 RNA polymerase is present which will recognize its cognate promoter (T7 promoter) and therefore initiate expression of the downstream reporter gene (GFP).

Modeling

Current simulation results

topside view, htProt=2h	3d surface plot, htProt=2h
simulation results for AND gate, different protein halflife periods top / bottom: output (z axis) is mRNA rate (∼PoPS) / reporter concentration (x/y: inputs, z: output, htProt: protein halflife period)

For every plot, 25 (left) / 100 (right) ODE simulations were performed, simulating a time period of 12h. The simulations have been run with 2h halflife period for proteins. Complete parameter allocation can be found in the matlab scripts as well as the system of ordinary differential equations (ODEs).

The input rates (PoPS) have been chosen in the range of mRNA transcription rate, which was estimated according to the following assumptions:

• E.coli cytoplasm volume is approximately 6.7*10^-16 l
• average number of mRNA molecules: 10
  → concentration_mRNA = 10/(6.7*10^-16 * 6.022*10²³) M = 0.0248 μM
• at equilibrum, mRNA rate and degredation balance each other. Assuming half life period of 30min for mRNA, the result is
  → rate_mRNA = concentration_mRNA * log(2)/30 μM/min = 5.7265e-04 μM/min

Amplifying/damping the input rates by small constant factors has influence on the qualitative outcome of the simulation.

• it is thus important to know how strong the input of the gate has to be.
• we can regulate this by choosing/designing the predecessor gate accordingly or
• by changing the ribosome binding sites to strengthen/weaken the input signal.

We accounted for this by adding restriction enzyme sites to the DNA, so that we can cut out current RBS to replace it by another of different strength.

The simulations show pretty much AND like behavior, but as mentioned, it is sensitive to the input strength. We want to find out about sensitivity more generally:

Sensitivity analysis

More (old) simulation results

simulation results / sensitivity analysis

Assembly procedure

Test procedure

Test results

Parts

[http://partsregistry.org/Part:BBa_J34100 BBa_J34100]

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