Team:Wash U/Biological Parts

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Revision as of 22:11, 20 July 2009

Parts

Component	Description	Part/Accession #	Base Pairs	Plasmid	Resistance	Well
RBS	Ribosomal Binding Site	BBa_B0034	12	pSB1A2	Ampicillin	plate 1, 2M
Cph8	Red Light sensor	BBa_I15010	2,238	pSB2K3	Kanamycin	N/A
RFP	Red Fluorescent Protein	BBa_J04051	720			N/A
OmpR (E. coli)	description	NP_417864.1	720	pSB1T3	Tetracycline	N/A
Terminator	Stops Transcription	BBa_B0015	129	pSB1AK3	Ampicillin and Kanamycin	plate 1, 23L
OmpR + Terminator	description	sequence	916	pany-amp	Ampicillin	synthesized
OmpC promoter	description	BBa_R0082	108	pSB1A2	Ampicillin	plate 1, 16K
puc B/A	description	sequence	375	pSB1A3	Ampicillin	N/A
puc B	description	YP_353390	156	?	?	N/A
puc A	description	YP_353391	165	?	?	N/A
OmpC promoter+BA	description	sequence	539	pany-kana	Kanamycin	synthesized
Light Response System	description	BBa_M30109	4,333	?	Ampicillin	N/A
TetR repressible	description	BBa_J13002	74	pSB1A2	Ampicillin	plate 1, 13B
Green Fluorescent Protein	Marker for characterization	BBa_E0240	876	pSB1A2	Ampicillin	plate 1, 12M

Plasmids

Plasmid	Description	Base Pairs	Resistance	Copy Number
pSB1A2	BioBrick Vector	2,079	Ampicillin	high
pSB1K3		2,206	Kanamycin	high
pSB1A3	Assembly plasmid	2,157	Ampicillin	high
pSB2K3		4,425	Kanamycin	variable
pSB1T3	Assembly plasmid	2,463	Tetracycline	high
pSB1AK3	Assembly Plasmid	3,189	Ampicillin and Kanamycin	high
pANY	Synthesized from Geneart
pRKCBC3			Tetracycline

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Characterization

The first part of our characterization begins with the puc promoter. The puc promoter is what turns on the entire system naturally in Rhodobacter sphaeroides. The puc promoter is what ultimately controls the number of LH2 light harvesting complexes, which is how our system will yield an increase in photosynthetic efficiency. It is important that we are able to compare the transcription rate of the puc promoter in the two systems so that we can determine exactly how much efficiency is gained by adding a red light sensor. By attaching Green Fluorescent Protein (GFP) to the promoter we can quantify the rate of transcription by measuring the emission of green light using a fluorescence spectrophotometer. We would expect to see more fluorescence with more transcription and vis versa.

The next step in our characterization of our synthetic red light response system is to analyze changes in phosphorylation of ompR in Rhodobacter sphaeroides. In our final system, we only want puc genes to be transcribed and expressed via halting the autophosphorylation of ompR, not simply the puc promoter as it naturally occurs. By placing the ompR coding region downstream of the red light sensor and upstream of a terminator our modified system controls expression of the puc genes by the red light sensor. It should be impossible for the puc promoter to directly cause the transcription of puc genes due to the terminator, but instead, transcription of the puc genes must be activated via decreasing the presence of phosphorylated ompR. The end target of ompR transcription is the ompC promoter, located directly upstream of the puc genes. Placing GFP on the ompC transcript will show how often the promoter is transcribed and how often ompR is phoshorylated.

Part three of our characterization measures the effectiveness of the red light sensor in downregulating the phosphorylation of ompR. This setup is identical to that of part two except we have introduced the red light sensor. Now, the rate of ompR autophosphorylation will be halted by binding to a domain on the light-activated EnvZ kinase analogue. GFP is still attached to the end product, the ompC promoter. By comparing the fluorescence of GFP in this scenario compared with the second scenario the decrease in rate of phosphorylation should be apparent due to the activity of the red light sensor.

This is the final construct that will be our actual functioning model in Rhodobacter sphaeroides. This finished product will be compared to the wild type over various intensities of light and cell culture densities can be compared to see which strain, the wild type or the modified strain with the red light sensor was more efficient in harvesting light with varying intensities.

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Modeling

pucBA Expression Model Diagram

pucBA Model Equations

pucBA Model Reactions

Test Simulation Output

Modeling the Gene Regulatory Network

Our group seeks to assess the optimality of the synthetic system that modulates pucB/A gene expression and LH2 complex assembly in Rhodobacter sphaeroides. Here we employ a mathematical model of this system to generate predictions about the behavior of the active system in response to light input. Features of the system that the model may help investigate include the time scale of response to light signals, the robustness of the system in response to fluctuations in light intensity, and the translation between changes in gene expression and the absorbance spectrum of the engineered cells.

Though the context of the model can extend back to the transcription of PrrA/B genes involved in integration oxygen and light signals, a preliminary testing model was developed using assumptions of certain initial conditions to isolate the light signal's effect. Since Cph8 and OmpR are located on the same transcript downstream of the puc promoter region, it was assumed that their associated protein and mRNA had already reached steady state concentrations, and the phosphorylation reaction had already reached steady state. Moreover, the concentrations of the factors were assumed to be equal at this state. The model whose diagram was constructed in the Simbiology Toolbox distributed by MathWorks details key reactions leading to the translation of the pucB/A genes. The reaction rate equation used for the lack of phosphorylation of OmpR when the light signal reaches Cph8 bound to OmpR is captured in a modified form of Michaelis-Menten kinetics. A logic function that corresponds to light ON/OFF (1/0) multiplies the maximum reaction rate in the numerator of the phosphorylation equation. Thus, the model assumes that no phosphorylation occurs by this mechanism in the presence of light. The OmpC promoter binding equation was based on the Hill Equation for an Activator(1).

Component characterization steps and literature searches are underway in order to obtain quantitative parameters for the reaction rates. In order to simulate behavior of the system, putative values were included that exaggerate true concentrations and time scales. OmpR was given an initial concentration normalized to one, and all other components were assumed insignificant initially to this value. An ideal light pulse was introduced at an instant and removed thirty simulation seconds later. From this rudimentary simulation it can be drawn that the nonlinearities of the phosphorylation and transcription factor binding kinetics effectively smooth the sharp light input. By design, the light switch ON yields no phosphorylation of OmpR and repression of the pucB/A genes which would give rise to LH2. Conversely, when left OFF, the concentration of pucB/A recovers and increases until the steady state determined by its translation and degradation rates.

References

1. Alon, Uri. Introduction to systems biology and the design principles of biological networks. Boca Raton, FL: Chapman & Hall, 2006.
2. Bower, James M. Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology). New York: M.I.T. PRESS, 2001.
3. System modeling in cellular biology from concepts to nuts and bolts. Cambridge, MA: MIT P, 2006.

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@@ Line 209: / Line 209: @@
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-:Though the context of the model can extend back to the transcription of PrrA/B genes involved in integration oxygen and light signals, a preliminary testing model was developed using assumptions of certain initial conditions to isolate the light signal's effect.  Since Cph8 and OmpR are located on the same transcript downstream of the puc promoter region, it was assumed that their associated protein and mRNA had already reached steady state concentrations.  Moreover, the concentrations of the factors were assumed to be equal at this state.  The model whose diagram was constructed in the Simbiology Toolbox distributed by MathWorks details key reactions leading to the translation of the pucB/A genes.  The reaction rate equation used for the phosphorylation of OmpR as a consequence of the light signal reaching Cph8 bound to OmpR is capture in a modified form of Michaelis-Menten kinetics.  A logic function that corresponds to light ON/OFF (1/0) multiplies the maximum reaction rate in the numerator.  Thus, the model assumes that no phosphorylation occurs by this mechanism in the absence of light.  The OmpC promoter binding equation was based on the Hill Equation for a Repressor (1).
+:Though the context of the model can extend back to the transcription of PrrA/B genes involved in integration oxygen and light signals, a preliminary testing model was developed using assumptions of certain initial conditions to isolate the light signal's effect.  Since Cph8 and OmpR are located on the same transcript downstream of the puc promoter region, it was assumed that their associated protein and mRNA had already reached steady state concentrations, and the phosphorylation reaction had already reached steady state.  Moreover, the concentrations of the factors were assumed to be equal at this state.  The model whose diagram was constructed in the Simbiology Toolbox distributed by MathWorks details key reactions leading to the translation of the pucB/A genes.  The reaction rate equation used for the lack of phosphorylation of OmpR when the light signal reaches Cph8 bound to OmpR is captured in a modified form of Michaelis-Menten kinetics.  A logic function that corresponds to light ON/OFF (1/0) multiplies the maximum reaction rate in the numerator of the phosphorylation equation.  Thus, the model assumes that no phosphorylation occurs by this mechanism in the presence of light.  The OmpC promoter binding equation was based on the Hill Equation for an Activator(1).
 <br><br><br>
-:Component characterization steps and literature searches are underway in order to obtain quantitative parameters for the reaction rates.  In order to simulate behavior of the system, putative values were included that exaggerate true concentrations and time scales.  OmpR was given an initial concentration normalized to one, and all other components were assumed insignificant initially to this value. An ideal light pulse was introduced at an instant and removed thirty simulation seconds later. From this rudimentary simulation it can be drawn that the nonlinearities of the phosphorylation and transcription factor binding kinetics effectively smooth the sharp light input.  By design, the light switch ON yields phosphorylation of OmpR and repression of the pucB/A genes which would give rise to LH2.  Conversely, when left OFF, the concentration of pucB/A recovers and increases until the steady state determined by its translation and degradation rates.
+:Component characterization steps and literature searches are underway in order to obtain quantitative parameters for the reaction rates.  In order to simulate behavior of the system, putative values were included that exaggerate true concentrations and time scales.  OmpR was given an initial concentration normalized to one, and all other components were assumed insignificant initially to this value. An ideal light pulse was introduced at an instant and removed thirty simulation seconds later. From this rudimentary simulation it can be drawn that the nonlinearities of the phosphorylation and transcription factor binding kinetics effectively smooth the sharp light input.  By design, the light switch ON yields no phosphorylation of OmpR and repression of the pucB/A genes which would give rise to LH2.  Conversely, when left OFF, the concentration of pucB/A recovers and increases until the steady state determined by its translation and degradation rates.
 <br><br><br>