Team:KULeuven/Project/Filter

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Filter

BioBricks

Components

T7 RNA polymerase, N-terminal green, C-terminal yellow

The filter, acting as a feedforward loop with AND-gate and therefor needs three specific components.

The first is a fast degrading primary messenger. For this, we used [http://partsregistry.org/Part:BBa_J23009 BBa_J23009], the RiboKey3d (more information [http://partsregistry.org/Featured_Parts:RNA-Lock-and-Key here]). As this is an mRNA molecule, degradation occurs quite fast (referentie).
The second component of the system is a stable secondary messenger, that is induced by the first one. We opted for T7 RNA polymerase ([http://partsregistry.org/Part:BBa_K145xxx BBa_K145xxx]). This molecule is much more stable than mRNA's, and modeling told us it was in fact too stable. So an UmuD derived N-terminal tag was added. Indeed, the crystal structure of T7 RNA polymerase showed catalytic activity at the C-terminus, which seems to be buried at the inside of the protein. The N-terminal is not catalytically active and also much more accessible. For more information on the UmuD-derived N-terminal tag, see the literature.
The third and last specific component is an AND-gate. To make such a gate, we placed de filter output signal under control of the T7 promoter ([http://partsregistry.org/Part:BBa_I712074 BBa_I712074]) and locked by the RiboLock3d ([http://partsregistry.org/Part:BBa_J23032 BBa_J23032]). This AND-gate as such is not a part of the filter mechanism, but serves as promoter and RBS of parts that are dependent on the filter.

Action

The components mentioned above now work in concert in what is called a coherent feedforward loop with AND-gate ([http://www.nature.com/ng/journal/v31/n1/abs/ng881.html reference]). Its action is particulary dependent on the above-mentioned differences in degradation time of the primary and secondary messenger. If background input signal is present, a small amount of primary messenger will result in a short amount of secondary messenger, but as a result of the time delay between the two, the primary signal will be degraded once the secondary signal is present. Because of this, the AND-gate will give no signal. Given a vast amount of input signal, the primary messenger will still be present once the secondary messenger is formed. This will lead to a nonzero signal from the AND-gate.

Indeed, production of the RiboKey3d will give rise to T7 RNA polymerase production. The T7 promoter and RiboLock3d together now form the AND-gate, giving transcription only when both the T7 RNA polymerase and RiboLock3d are present together.

For more information on the exact kinetics and dynamics of this system, see the modeling page (link).

@@ Line 1: / Line 1: @@
 {{:Team:KULeuven/Tools/Header}}
-<div style="float: right;">[[Image:pictogram_filter.png|70px]]</div>
+<div style="float: right;">[[Image:pictogram_filter.png|120px]]</div>
 ==Filter==
+===BioBricks===
 [[Image:Filter_BioBrick.jpg|center]]
-The filter mechanism makes use of a coherent feedforward loop with and gate ('''reference'''). For this system, we thus needed a primary messenger, which gives a signal that leads to a secondary messenger, and that is fast degrading. The secondary messenger is a signal that is more stable in time. They cooperate together in the AND-gate to result in another signal. This last signal is the output of the filter.
+===Components===
+[[Image:T7RNAP.png|290px|right|T7 RNA polymerase, N-terminal green, C-terminal yellow]]The filter, acting as a feedforward loop with AND-gate and therefor needs three specific components.
+* The ''first'' is a fast degrading primary messenger. For this, we used [http://partsregistry.org/Part:BBa_J23009 '''BBa_J23009'''], the RiboKey3d (more information [http://partsregistry.org/Featured_Parts:RNA-Lock-and-Key here]). As this is an mRNA molecule, degradation occurs quite fast ('''referentie''').
+* The ''second'' component of the system is a stable secondary messenger, that is induced by the first one. We opted for T7 RNA polymerase ([http://partsregistry.org/Part:BBa_K145xxx '''BBa_K145xxx''']). This molecule is much more stable than mRNA's, and modeling told us it was in fact too stable. So an UmuD derived N-terminal tag was added. Indeed, the crystal structure of T7 RNA polymerase showed catalytic activity at the C-terminus, which seems to be buried at the inside of the protein. The N-terminal is not catalytically active and also much more accessible. For more information on the UmuD-derived N-terminal tag, see the [https://2008.igem.org/Team:KULeuven/Literature literature].
+* The ''third'' and last specific component is an AND-gate. To make such a gate, we placed de filter output signal under control of the T7 promoter ([http://partsregistry.org/Part:BBa_I712074 '''BBa_I712074''']) and locked by the RiboLock3d ([http://partsregistry.org/Part:BBa_J23032 '''BBa_J23032''']). This AND-gate as such is not a part of the filter mechanism, but serves as promoter and RBS of parts that are dependent on the filter.
-Among the fast degrading molecules in a bacterial cell are mRNA's. We used the key-lock system as a primary messenger. Upon input signal, '''BBa_J23009''' is transcribed and RiboKey3d is produced. This mRNA has a relatively short lifetime ('''referentie naar parameters van de ir's'''). This key can unlock the RiboLock3d, which is part '''BBa_J23032'''.
+===Action===
+[[Image:filter_1.jpg|140px|left]]The components mentioned above now work in concert in what is called a coherent feedforward loop with AND-gate ([http://www.nature.com/ng/journal/v31/n1/abs/ng881.html reference]). Its action is particulary dependent on the above-mentioned differences in degradation time of the primary and secondary messenger. If background input signal is present, a small amount of primary messenger will result in a short amount of secondary messenger, but as a result of the time delay between the two, the primary signal will be degraded once the secondary signal is present. Because of this, the AND-gate will give no signal. Given a vast amount of input signal, the primary messenger will still be present once the secondary messenger is formed. This will lead to a nonzero signal from the AND-gate.
-Under the constitutive promoter '''BBa_I23110''', and locked by RiboLock3d, we find '''BBa_K145xxx''', a T7 RNA polymerase with UmuD-derived tag. This T7 RNA polymerase is a protein, and thus much more stable than mRNA. So stable, that modeling proved it was too stable. So a tag has been added to make it degrade faster. A C-terminal LVA tag would probably not have worked, as the C-terminal of T7 RNA polymerase is part of the catalytic region ('''figuur'''). An N-terminal tag, however, is the solution to this problem. More information on this N-terminal, UmuD derived tag can be found here ('''link''').
+Indeed, production of the RiboKey3d will give rise to T7 RNA polymerase production. The T7 promoter and RiboLock3d together now form the AND-gate, giving transcription only when both the T7 RNA polymerase and RiboLock3d are present together.
-We see now, that production of the RiboLock3d will give rise to T7 RNA polymerase production, and that the requirements concerning the lifetime of the messenger molecules are fulfilled. The T7 promoter and RiboLock3d together now form the AND-gate, giving transcription only when both the T7 RNA polymerase and RiboLock3d are present together.
 For more information on the exact kinetics and dynamics of this system, see the modeling page ('''link''').