Difference between revisions of "Part:BBa K1895997"

Revision as of 19:00, 27 September 2016

Blue light sensitive 'LDR'

In addition to designing an arabinose controlled variable resistor for our iGEM work we also investigated alternative methods of controlling resistance that are found in common electronic components. One such example is the light dependent resistor, or LDR. Our biological LDR is based on the same scheme as our arabinose controlled resistor. That is, using E. coli to vary the amount of free ions in an electrolyte. Ion uptake will be controlled by the expression of a protein, smtA which is a metallothionein capable of binding to heavy metal ions like cadmium (II), Zinc (II) and Copper (II).

For this LDR we will be using a blue light sensitive protein system. In this scheme the production of SmtA which affects the resistivity is placed under the control of of a FixJ-P (phosphorylated FixJ) promoter. This allows the protein production to be regulated by blue light through a series of reactions with the FixJ response regulator protein YF1.

We have also designed a red light sensitive LDR using OmpR.

Usage and Biology

In the absence of light, YF1 undergoes autophosphorylation to produce YF1-P which can phosphorylate FixJ. This in turn activates the transcription of the downstream protein. In this case that is SmtA. Thus, in the presence of light SmtA is not produced and so conductivity does not change, whilst in the absence of light SmtA is produced resulting in a decrease in resistance.

File:Https://static.igem.org/mediawiki/2016/7/7f/T--Newcastle--YFP FIXJ.png

caption Diagram showing how FixJ and YF1 interact in blue light.

This behaviour is the inverse of an electrical light dependent resistor where resistance increases with light intensity. To mimic this behaviour using biological circuits we would place an inverter before the FixK2 promoter (which is activated by FixJ-P). The inverter is constructed by placing the desired output, here SmtA, under the control of a lambda cl regulated promoter (BBa_R0051). As lambda cl represses the promoter, having the lambda cl protein itself produced under control of FixK2 promoter inverts the system. This results in SmtA being produced in the presence of light rather than the absence thereof. BBa_K592020 is an example of a part that uses this technique.

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 <partinfo>BBa_K1895997 short</partinfo>
-In addition to designing an [https://parts.igem.org/Part:BBa_K1895999 arabinose controlled variable resistor] for our iGEM work we also investigated alternative methods of controlling resistance that are found in common electronic components. One such example is the light dependent resistor, or LDR. Our biological LDR is based on the same scheme as our arabinose controlled resistor. That is, using E. coli to vary the amount of free ions in an electrolyte. Ion uptake will be controlled by the expression of a protein, [https://parts.igem.org/Part:BBa_K1895998 smtA] which is a metallothionein capable of binding to heavy metal ions like cadmium (II), Zinc (II) and Copper (II).
+In addition to designing an arabinose controlled variable resistor for our iGEM work we also investigated alternative methods of controlling resistance that are found in common electronic components. One such example is the light dependent resistor, or LDR. Our biological LDR is based on the same scheme as our arabinose controlled resistor. That is, using E. coli to vary the amount of free ions in an electrolyte. Ion uptake will be controlled by the expression of a protein, smtA which is a metallothionein capable of binding to heavy metal ions like cadmium (II), Zinc (II) and Copper (II).
-For this LDR we will be using the red light detection system from the [https://parts.igem.org/Coliroid Colliroid] project. In this scheme the production of SmtA which affects the resistivity is placed under the control of the OmpF upstream promoter ([https://parts.igem.org/Part:BBa_R0084 BBa_R0084]). Through the addition of a genetic invertor circuit we hope this part will form a system system where this promoter is repressed in the dark and has increased transcription under (red) light. This would the device to mimic the behaviour of a traditional electronic LDR whereby resistance is decreased in the light and increased in the dark.
+For this LDR we will be using a blue light sensitive protein system. In this scheme the production of SmtA which affects the resistivity is placed under the control of of a FixJ-P (phosphorylated FixJ) promoter. This allows the protein production to be regulated by blue light through a series of reactions with the FixJ response regulator protein YF1.
-We have also designed a [https://parts.igem.org/Part:BBa_K1895997 blue light sensitive LDR] using FixJ and YF1.
+We have also designed a [https://parts.igem.org/Part:BBa_K1895996 red light sensitive LDR] using OmpR.
 <!-- Add more about the biology of this part here -->
 ===Usage and Biology===
-The OmpF promoter is repressed by phosphorylated OmpR, OmpR-P. In normal conditions the E. coli cell contains free OmpR which can be phosphorylated by expression of a protein with an EnvZ domain. One such protein is the fusion protein, Cph8 ([https://parts.igem.org/wiki/index.php/Part:BBa_I15010 BBa_I15010]).
+In the absence of light, YF1 undergoes autophosphorylation to produce YF1-P which can phosphorylate FixJ. This in turn activates the transcription of the downstream protein. In this case that is SmtA. Thus, in the presence of light SmtA is not produced and so conductivity does not change, whilst in the absence of light SmtA is produced resulting in a decrease in resistance.
-In order for the light responsive domain of the fusion protein cph8 to sense red light the formation of a chromophore is required this is done by the production of two proteins, ho1 and PcyA together with the cph8. In our system these will be constitutively expressed to create the red light sensor.
+[[File:https://static.igem.org/mediawiki/2016/7/7f/T--Newcastle--YFP_FIXJ.png|frame|none|alt=|caption Diagram showing how FixJ and YF1 interact in blue light.]]
-In the dark the fusion protein, Cph8 phosphorylates OmpR and thereby prevents SmtA production as the OmpF promoter is repressed. This increases resistance. In the light, the light responsive domain Cph1 inhibits the activity of the EnvZ is prevented from phosphorylating OmpR and therefore allows SmtA production and decreased resistance.
+This behaviour is the inverse of an electrical light dependent resistor where resistance increases with light intensity. To mimic this behaviour using biological circuits we would place an inverter before the FixK2 promoter (which is activated by FixJ-P). The inverter is constructed by placing the desired output, here SmtA, under the control of a lambda cl regulated promoter ([https://parts.igem.org/wiki/index.php/Part:BBa_R0051 BBa_R0051]). As lambda cl represses the promoter, having the lambda cl protein itself produced under control of FixK2 promoter inverts the system. This results in SmtA being produced in the presence of light rather than the absence thereof. [https://parts.igem.org/wiki/index.php/Part:BBa_K592020 BBa_K592020] is an example of a part that uses this technique.
-Through the addition of a basic genetic inverter circuit it would be possible to swap this behaviour so that the resistance decreases in the dark and increases in the light depending on the desired behaviour of your system.
-'''Please note''' that this will only work in E. coli which are naturally deficient in EnvZ.
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