Team:NYMU-Taipei/Modeling/Ethanol

From 2013.igem.org

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Latest revision as of 02:39, 29 October 2013

National Yang Ming University

1 Ethanol model

Ethanol model

Background

AhpCp promoter is an oxyR-activated promoter. Behind AhpCp promoter are several terminals to inhibit T7 polymerase-producing gene from being opened easily ; namely, it will not open until the concentration of oxyR is high enough, which means the bees are facing disastrous infection. Once oxyR concentration overcomes the threshold and conquers the terminal obstacles, T7 polymerase is produced and will bind to T7 promoter, which is a specific promoter binding only to T7 polymerase. Behind the T7promoter are enzyme PDC and ADH-producing genes, which will convert pyruvate to ethanol, and thus kill spores of Nosema Ceranae. Pathway of ethanol

PDC= pyruvate decarboxylase; ADH= Acetaldehyde

Objectives

To simulate how many terminal do we need as a threshold to have the T7 polymerase-producing gene open at a proper 　　time.
To simulate the threshold concentration of oxyR to conquer the terminal.
To determine the time of opening to see if the circuit could open in time (useful).

System

It is assumed that the dose of pyruvate is sufficient in bees’ body through enough proliferation/copy number of Beecoli; the possibility of RNApolymerase skipping terminators is assumed to be proportional to time span; equilibrium between pyruvate and acetaldehyde is dominantly rightward, while equilibrium between acetaldehyde and ethanol is bidirectional; concentration of ethanol will not easily decrease and will sustain for a period of time.

As for how we measure promoter strengths, we choose PoPS, which is the rate of RNA polymerase binding to the DNA and trigger the transcription of the certain gene.

Equation1

\frac{d[mRNA_T_7]}{dt}=\frac{ [ROSoxyR]^{nROSoxyR} }{ KdROSoxyR^{nROSoxyR}+[ROSoxyR]^{nROSoxyR} }\times{PoPSAhpCp}\times\frac{N}{V}\times{a}+\frac{ [T7]^{nT7} }{ KdT7^{nT7}+[T7]^{nY7} }\times{PoPST7}\times\frac{N}{V}-KdegmRNA\times[mRNAPDC]

KdROSoxyR = dissociation constant of ROSoxyR
n ROSoxyR = Hill coefficient of ROSoxyR
PoPSAhpCp = promoter strength of AhpCp
KdT7 = dissociation constant of T7
NT7 = Hill coefficient of T7
PoPST7 = promoter strength of T7 promoter
KdegmRNA = degrading constant of sensor promoter mRNA
N = number of plasmid in a single cell
V = volume of a cell

more1

Equation2

\frac{d[protein of T7 polymerase ]}{dt}=RBS\times{PoPSAhpCp}\times\frac{N}{V}\times{a}\times{\frac{ [ROSoxyR]^{n1} }{ [Kd]^{n1}+[ROSoxyR]^{n1} }}}+PoPST7\times{T7 effect}\times\frac{N}{V}-{KdegT7}\times{T7}

PoPSAhpCp = promotor strength of AhpCp promoter
PoPST7 = promoter strength of T7 promoter
RBS = binding site strength
kdegT7 = degrading constant of T7 polymerase
a = posibility of conquering the threshold concentration
n1 = Hill coefficient of ROSoxyR (complex of ROS+oxyR)
N = number of plasmid in a single cell
V = volume of a cell

The aim of the equation is to know the threshold concentration of oxyR to conquer the terminal and when T7 polymerase can reach the required concentration to activate T7 promoter.

more2

Equation3

\frac{d[mRNA_P_D_C]}{dt}=\frac{ [T7]^{nT7} }{ KdT7^{nT7}+[T7]^{nY7} }\times{PoPST7}\times\frac{N}{V}-KdegmRNA\times[mRNAPDC]

KdT7 = dissociation constant of T7
NT7 = Hill coefficient of T7
PoPST7 = promoter strength of T7 promoter
KdegmRNA = degrading constant of sensor promoter mRNA
N = number of plasmid in a single cell
V = volume of a cell

The aim of the equation is to know mRNA of enzyme PDC production rate and when it can reach the level to translate enough PDC.

more3

Equation4

\frac{d[PDC]}{dt}=RBS\times{PoPST7effect}\times\frac{N}{V}-{kdegPDC}\times[PDC}]

RBS = binding site strength
PoPST7 = promoter strength of T7 promoter
N = number of plasmid in a single cell
V = volume of a cell
KdegPDC = degrading constant of PDC (pyruvate decarboxylase)

The aim of the equation is to know PDC production rate and when it can reach the concentration of ethanol pathway equilibrium.

more4

Equation5

\frac{d[mRNA_A_D_H]}{dt}=\frac{ [T7]^{nT7} }{ KdT7^{nT7}+[T7]^{nY7} }\times{PoPST7}\times\frac{N}{V}-KdegmRNA\times[mRNAADH]

KdT7 = dissociation constant of T7
NT7 = Hill coefficient of T7
PoPST7 = promoter strength of T7 promoter
KdegmRNA = degrading constant of sensor promoter mRNA
N = number of plasmid in a single cell
V = volume of a cell

more5

Equation6

\frac{d[ADH]}{dt}=RBS\times{PoPST7effect}\times\frac{N}{V}-{KdegADH}\times[ADH]}

RBS = binding site strength
PoPST7 = promoter strength of T7 promoter
N = number of plasmid in a single cell
V = volume of a cell
KdegADH = degrading constant of ADH (Acetaldehyde)

The aim of the equation is to know ADH production rate and when it can reach the concentration of ethanol pathway equilibrium.

more6

Equation7

\frac{d[ethanol]}{dt}=Kpyruvateacetaldehyde\times{Kacetaldehydeethanol}\times\frac{[PDC]^{nPDC} }{ KdPDC^{nPDC}+[PDC]^{nPDC}}\times\frac{[ADH]^{nADH} }{ KdADH^{nADH}+[ADH]^{nADH}}-Km\times\frac{[ADH]^{nADH}}{ KdADH^{nADH}+[ADH]^{nADH}}\times[ethonal]

Kpyruvateacetaldehyde = pyruvate→acetaldehyde reaction rate constant
Kacetaldehydeethanol = acetaldehyde→ethanol reaction rate constant
Km = ethanol→acetaldehyde reaction rate constant
KdADH = dissociation constant of ADH

The aim of the equation is to know ethanol production rate and when it can reach the concentration to kill the spores of Nosema Ceranae.

more7

Results

Figure1:this figure shows that after adding several terminators and T7 polymerase mechanism, we successfully build a time delay as well as a boom of PDC and ADH enzymes to attain our goal-ethanol production can reach its effective concentration(0.0005M) after 80 hours of Nosema infection.

Figure2:This picture is a control group model. It shows that without Nosema infection (ROS concentration=0), ethanol production is low enough to be ignored. As a result, the bee will not be harmed by ethanol.

Figure3: ethanol production turning off after Nosema is killed (step 1)

Figure4: ethanol production turning off after Nosema is killed (step 2)

Figure3, Figure4: The two pictures above show that if Nosema is killed by kill protein effectively, the ethanol production will soon be turned off (will not reach the effective concentration and decrease to a neglectable low level).

Discussion

Figures show that after sensor AhpCp is activated, ethanol reaches its effective concentration 72-80 hours after the infection, which is the time in Nosema lifecycle when bees carrying Nosema spores become incurable and infectious to other bees), In other words, this means if killing protein targeting at Nosema wasn't produced or functioning properly (click here to see the previous model for killing protein), Beecoli turns against the sick bee to kill it within 3-4days.

To make ethanol production on time, we adjusted the expression level of OxyR (click here for more information), the terminators in front of the desired gene, and the promoter strength for independent production of PDC and ADH(click here for detailed information about circuit design).

Circuit Design and Improvements

To postpone ethanol production until it is needed, we adjusted the terminators’ effect in terms of possibility (click here to see the ODE). Results show that there’s a short lag (8-10 hours) between invasion sensing and ethanol production rather than a long one expected. This means that possibility isn’t a sensitive parameter and therefore adding terminator is not an efficient way to manipulate timing when ethanol is produced. Nevertheless, these terminators still has the function of preventing leakage of T7 polymerase production (click here for detailed information about circuit design). By adjusting the parameters in the ODEs given, we found that the nature of T7 transcription is key to adjusting ethanol production time. If postponing ethanol production is needed, a T7 promoter with lower promoter strength and T7 polymerase with higher degrading rate should be chosen

Parameters

Model	Parameter	Description	Value	Unit	Reference
Ethanol	pyruvate	Initial concentration of pyruvate in MG1655	1.18 x 2	g/L	Expression of pyruvate carboxylase enhances succinate production in Escherichia coli without affecting glucose uptake
	nPDC	Hill coefficient of PDC	2.1	-	Purification, characterization and cDNA sequencing of pyruvate decarboxylase Zygosaccharomyces biporus
	nADH	Hill coefficient of ADH	at high pH values, 30◦C, n=1; at low temperature, n=3	-	Evidence for co-operativity in coenzyme binding to tetrameric Sulfolobus solfataricus alcohol dehydrogenase and its structural basis: ﬂuorescence, kinetic and structural studies of the wild-type enzyme and non-co-operative N249Y mutant

Retrieved from "http://2013.igem.org/Team:NYMU-Taipei/Modeling/Ethanol"

@@ Line 1: / Line 1: @@
 {{:Team:NYMU-Taipei/Header}}
-==Ethanol model==
+=Ethanol model=
-===Background:===
+==Background==
-TrxC promoter is an oxyR-activated promoter. Behind trxC promoter are several terminals to inhibit T7 polymerase-producing gene from being opened easily ; namely, it will not open until the concentration of oxyR is high enough, which means the bees are facing disastrous infection. Once oxyR concentration overcomes the threshold and conquers the terminal obstacles, T7 polymerase is produced and will bind to T7 promoter, which is a specific promoter binding only to T7 polymerase. Behind the T7promoter are enzyme PDC and ADH-producing genes, which will convert pyruvate to ethanol, and thus kill spores of ''Nosema Ceranae''.
+AhpCp promoter is an oxyR-activated promoter. Behind AhpCp promoter are several terminals to inhibit T7 polymerase-producing gene from being opened easily ; namely, it will not open until the concentration of oxyR is high enough, which means the bees are facing disastrous infection. Once oxyR concentration overcomes the threshold and conquers the terminal obstacles, T7 polymerase is produced and will bind to T7 promoter, which is a specific promoter binding only to T7 polymerase. Behind the T7promoter are enzyme PDC and ADH-producing genes, which will convert pyruvate to ethanol, and thus kill spores of ''Nosema Ceranae''.
-====Pathway of ethanol:====
+'''Pathway of ethanol'''
 [[File:NYMU_eth.png]]
+[[File:NYMU_ethanol pathway.png]]
 PDC= pyruvate decarboxylase; ADH= Acetaldehyde
-===Aims===
+==Objectives==
 <br>
-.To simulate how many terminal do we need as a threshold to have the T7 polymerase-producing gene open at a proper 　　time.<br>
+#To simulate how many terminal do we need as a threshold to have the T7 polymerase-producing gene open at a proper 　　time.<br>
-.To simulate the threshold concentration of oxyR to conquer the terminal.<br>
+#To simulate the threshold concentration of oxyR to conquer the terminal.<br>
-.To determine the time of opening to see if the circuit could open in time (useful).<br>
+#To determine the time of opening to see if the circuit could open in time (useful).<br>
-<br>
+==System==
-　　As for how we measure promoter strengths, we choose PoPS, which is the rate of RNA polymerase binding to the DNA and trigger the transcription of the certain gene.<br>
+It is assumed that the dose of pyruvate is sufficient in bees’ body through enough proliferation/copy number of Beecoli; the possibility of RNApolymerase skipping terminators is assumed to be proportional to time span; equilibrium between pyruvate and acetaldehyde is dominantly rightward, while equilibrium between acetaldehyde and ethanol is bidirectional; concentration of ethanol will not easily decrease and will sustain for a period of time.<br>
-　　It is assumed that the dose of pyruvate is sufficient in bees’ body through enough proliferation/copy number of Beecoli; the possibility of RNApolymerase skipping terminators is assumed to be proportional to time span; equilibrium between pyruvate and acetaldehyde is dominantly rightward, while equilibrium between acetaldehyde and ethanol is bidirectional; concentration of ethanol will not easily decrease and will sustain for a period of time.<br>
+As for how we measure promoter strengths, we choose PoPS, which is the rate of RNA polymerase binding to the DNA and trigger the transcription of the certain gene.<br>
-===Equation1:===
+'''Equation1'''
 <html>
 <div lang="latex" class="equation">
-\frac{d[mRNAT7]}{dt}=\frac{ [ROSoxyR]^{nROSoxyR} }{ KdROSoxyR^{nROSoxyR}+[ROSoxyR]^{nROSoxyR} }\times{PoPStrxC}\times\frac{N}{V}\times{a}+\frac{ [T7]^{nT7} }{ KdT7^{nT7}+[T7]^{nY7} }\times{PoPST7}\times\frac{N}{V}-KdegmRNA\times[mRNAPDC]
+\frac{d[mRNA_T_7]}{dt}=\frac{ [ROSoxyR]^{nROSoxyR} }{ KdROSoxyR^{nROSoxyR}+[ROSoxyR]^{nROSoxyR} }\times{PoPSAhpCp}\times\frac{N}{V}\times{a}+\frac{ [T7]^{nT7} }{ KdT7^{nT7}+[T7]^{nY7} }\times{PoPST7}\times\frac{N}{V}-KdegmRNA\times[mRNAPDC]
 </div>
 </html>
 <br>
-.KdROSoxyR = dissociation constant of ROSoxyR<br>
+#KdROSoxyR = dissociation constant of ROSoxyR<br>
-.n ROSoxyR = Hill coefficient of ROSoxyR<br>
+#n ROSoxyR = Hill coefficient of ROSoxyR<br>
-.PoPStrxC = promoter strength of trxC<br>
+#PoPSAhpCp = promoter strength of AhpCp<br>
-.KdT7 = dissociation constant of T7<br>
+#KdT7 = dissociation constant of T7<br>
-.NT7 = Hill coefficient of T7<br>
+#NT7 = Hill coefficient of T7<br>
-.PoPST7 = promoter strength of T7 promoter<br>
+#PoPST7 = promoter strength of T7 promoter<br>
-.KdegmRNA = degrading constant of sensor promoter mRNA<br>
+#KdegmRNA = degrading constant of sensor promoter mRNA<br>
-.N = number of plasmid in a single cell<br>
+#N = number of plasmid in a single cell<br>
-.V = volume of a cell<br>
+#V = volume of a cell<br>
+[[more1]]
-===Equation2:===
+'''Equation2'''
 <html>
 <div lang="latex" class="equation">
-\frac{d[protein of T7 polymerase ]}{dt}=RBS\times{PoPStrxC}\times\frac{N}{V}\times{a}\times{\frac{ [ROSoxyR]^{n1} }{ [Kd]^{n1}+[ROSoxyR]^{n1} }}}+PoPST7\times{T7 effect}\times\frac{N}{V}-{KdegT7}\times{T7}
+\frac{d[protein of T7 polymerase ]}{dt}=RBS\times{PoPSAhpCp}\times\frac{N}{V}\times{a}\times{\frac{ [ROSoxyR]^{n1} }{ [Kd]^{n1}+[ROSoxyR]^{n1} }}}+PoPST7\times{T7 effect}\times\frac{N}{V}-{KdegT7}\times{T7}
 </div>
 </html>
 <br>
-.PoPStrxC = promotor strength of trxC promoter<br>
+#PoPSAhpCp = promotor strength of AhpCp promoter<br>
-.PoPST7 = promoter strength of T7 promoter<br>
+#PoPST7 = promoter strength of T7 promoter<br>
-.RBS = binding site strength<br>
+#RBS = binding site strength<br>
-.kdegT7 = degrading constant of T7 polymerase<br>
+#kdegT7 = degrading constant of T7 polymerase<br>
-.a = posibility of conquering the threshold concentration<br>
+#a = posibility of conquering the threshold concentration<br>
-.n1 = Hill coefficient of ROSoxyR (complex of ROS+oxyR)<br>
+#n1 = Hill coefficient of ROSoxyR (complex of ROS+oxyR)<br>
-.N = number of plasmid in a single cell <br>
+#N = number of plasmid in a single cell <br>
-.V = volume of a cell<br>
+#V = volume of a cell<br>
 '''The aim of the equation is to know the threshold concentration of oxyR to conquer the terminal and when T7 polymerase can reach the required concentration to activate T7 promoter.'''
+[[more2]]
-===Equation3:===
+'''Equation3'''
 <html>
 <div lang="latex" class="equation">
-\frac{d[mRNAPDC]}{dt}=\frac{ [T7]^{nT7} }{ KdT7^{nT7}+[T7]^{nY7} }\times{PoPST7}\times\frac{N}{V}-KdegmRNA\times[mRNAPDC]
+\frac{d[mRNA_P_D_C]}{dt}=\frac{ [T7]^{nT7} }{ KdT7^{nT7}+[T7]^{nY7} }\times{PoPST7}\times\frac{N}{V}-KdegmRNA\times[mRNAPDC]
 </div>
 </html>
 <br>
-.KdT7 = dissociation constant of T7<br>
+#KdT7 = dissociation constant of T7<br>
-.NT7 = Hill coefficient of T7<br>
+#NT7 = Hill coefficient of T7<br>
-.PoPST7 = promoter strength of T7 promoter<br>
+#PoPST7 = promoter strength of T7 promoter<br>
-.KdegmRNA = degrading constant of sensor promoter mRNA<br>
+#KdegmRNA = degrading constant of sensor promoter mRNA<br>
-.N = number of plasmid in a single cell<br>
+#N = number of plasmid in a single cell<br>
-.V = volume of a cell<br>
+#V = volume of a cell<br>
 '''The aim of the equation is to know mRNA of enzyme PDC production rate and when it can reach the level to translate enough PDC.'''
+[[more3]]
-===Equation4:===
+'''Equation4'''
 <html>
 <div lang="latex" class="equation">
@@ Line 81: / Line 86: @@
 </html>
 <br>
-.RBS = binding site strength<br>
+#RBS = binding site strength<br>
-.PoPST7 = promoter strength of T7 promoter<br>
+#PoPST7 = promoter strength of T7 promoter<br>
-.N = number of plasmid in a single cell<br>
+#N = number of plasmid in a single cell<br>
-.V = volume of a cell<br>
+#V = volume of a cell<br>
-.KdegPDC = degrading constant of PDC (pyruvate decarboxylase)<br>
+#KdegPDC = degrading constant of PDC (pyruvate decarboxylase)<br>
 '''The aim of the equation is to know PDC production rate and when it can reach the concentration of ethanol pathway equilibrium.'''
+[[more4]]
-===Equation5:===
+'''Equation5'''
 <html>
 <div lang="latex" class="equation">
-\frac{d[mRNAADH]}{dt}=\frac{ [T7]^{nT7} }{ KdT7^{nT7}+[T7]^{nY7} }\times{PoPST7}\times\frac{N}{V}-KdegmRNA\times[mRNAADH]
+\frac{d[mRNA_A_D_H]}{dt}=\frac{ [T7]^{nT7} }{ KdT7^{nT7}+[T7]^{nY7} }\times{PoPST7}\times\frac{N}{V}-KdegmRNA\times[mRNAADH]
 </div>
 </html>
 <br>
-.KdT7 = dissociation constant of T7<br>
+#KdT7 = dissociation constant of T7<br>
-.NT7 = Hill coefficient of T7<br>
+#NT7 = Hill coefficient of T7<br>
-.PoPST7 = promoter strength of T7 promoter<br>
+#PoPST7 = promoter strength of T7 promoter<br>
-.KdegmRNA = degrading constant of sensor promoter mRNA<br>
+#KdegmRNA = degrading constant of sensor promoter mRNA<br>
-.N = number of plasmid in a single cell<br>
+#N = number of plasmid in a single cell<br>
-.V = volume of a cell<br>
+#V = volume of a cell<br>
+[[more5]]
-===Equation6:===
+'''Equation6'''
 <html>
 <div lang="latex" class="equation">
@@ Line 113: / Line 120: @@
 </html>
 <br>
-.RBS = binding site strength<br>
+#RBS = binding site strength<br>
-.PoPST7 = promoter strength of T7 promoter<br>
+#PoPST7 = promoter strength of T7 promoter<br>
-.N = number of plasmid in a single cell<br>
+#N = number of plasmid in a single cell<br>
-.V = volume of a cell<br>
+#V = volume of a cell<br>
-.KdegADH = degrading constant of ADH (Acetaldehyde)<br>
+#KdegADH = degrading constant of ADH (Acetaldehyde)<br>
 '''The aim of the equation is to know ADH production rate and when it can reach the concentration of ethanol pathway equilibrium.'''
+[[more6]]
-===Equation7:===
+'''Equation7'''
 <html>
 <div lang="latex" class="equation">
@@ Line 130: / Line 138: @@
 </html>
 <br>
-.Kpyruvateacetaldehyde = pyruvate→acetaldehyde reaction rate constant<br>
+#Kpyruvateacetaldehyde = pyruvate→acetaldehyde reaction rate constant<br>
-.Kacetaldehydeethanol = acetaldehyde→ethanol reaction rate constant<br>
+#Kacetaldehydeethanol = acetaldehyde→ethanol reaction rate constant<br>
-.Km = ethanol→acetaldehyde reaction rate constant<br>
+#Km = ethanol→acetaldehyde reaction rate constant<br>
-.KdADH = dissociation constant of ADH<br>
+#KdADH = dissociation constant of ADH<br>
-'''The aim of the equation is to know ethanol production rate and when it can reach the concentration to kill the spores of Nosema Ceranae.'''
+'''The aim of the equation is to know ethanol production rate and when it can reach the concentration to kill the spores of ''Nosema'' Ceranae.'''
+[[more7]]
-==Explanation:==
-<html>
-<div lang="latex" class="equation">
-\frac{d[mRNAT7]}{dt}=\frac{ [ROSoxyR]^{nROSoxyR} }{ KdROSoxyR^{nROSoxyR}+[ROSoxyR]^{nROSoxyR} }\times{PoPStrxC}\times\frac{N}{V}\times{a}+\frac{ [T7]^{nT7} }{ KdT7^{nT7}+[T7]^{nY7} }\times{PoPST7}\times\frac{N}{V}-KdegmRNA\times[mRNAPDC]
-</div>
-</html>
-<br>
-　　PoPS represents the promoter strength which is measured by the rate of RNApolymerase binding to the starting site of DNA transcription; <html><span lang="latex">\frac{ [molecule]^{nmolecule} }{ Kdmolecule^{nmolecule}+[molecule]^{nmolecule} }</span></html> represents the hill effect of transcription factors (either activators or repressors) to the promoter.<br>
-In this equation, the production rate of mRNAT7 is composed of both synthesizing rate and degrading rate. <html><span lang="latex">\frac{ [ROSoxyR]^{nROSoxyR} }{ KdROSoxyR^{nROSoxyR}+[ROSoxyR]^{nROSoxyR} }\times{PoPStrxC}\times\frac{N}{V}\times{a}</span></html> represents how activator ROSoxyR influences promoter trxC on synthesizing mRNAT7 and how thresholds behind the promoter blocks the synthesis of mRNAT7 (the possibility of RNA polymerase to skip thresholds right after trxC promoter is shown in probability);<html><span lang="latex">\frac{ [T7]^{nT7} }{ KdT7^{nT7}+[T7]^{nY7} }\times{PoPST7｝\times\frac{N}{V}</span></html>represents how T7 polymerase influences promoter T7 on synthesizing mRNAT7; <html><span lang="latex">-KdegmRNA\times[mRNAT7]</span></html> represents the natural degrading rate of mRNAT7.<br>
+==Results==
-<html>
+[[File:NYMU_ethanol normal .jpg]]
-<div lang="latex" class="equation">
-\frac{d[protein of T7 polymerase ]}{dt}=RBS\times{PoPStrxC}\times\frac{N}{V}\times{a}\times{\frac{ [ROSoxyR]^{n1} }{ [Kd]^{n1}+[ROSoxyR]^{n1} }}}+PoPST7\times{T7 effect}\times\frac{N}{V}-{KdegT7}\times{T7}
-</div>
-</html>
-In this equation, the production rate of T7 polymerase is composed of both synthesizing rate and degrading rate. RBS represents ribosome binding site strength, which is the affinity of ribosome to the starting site of mRNA; PoPS represents the promoter strength and <html><span lang="latex">\frac{[molecule]^{nmolecule}}{ Kdmolecule^{nmolecule}+[molecule]^{nmolecule} }</span></html>, as mentioned above, represents the hill effect of transcription factors to the promoter.
+Figure1:this figure shows that after adding several terminators and T7 polymerase mechanism, we successfully build a time delay as well as a boom of PDC and ADH enzymes to attain our goal-ethanol production can reach its effective concentration(0.0005M) after 80 hours of ''Nosema'' infection.
-For the section of the equation,<html><span lang="latex">\RBS\times{PoPStrxC}\times\frac{N}{V}\times{a}\times{\frac{ [ROSoxyR]^{n1} }{ [Kd]^{n1}+[ROSoxyR]^{n1} }}}</span></html> represents the synthesizing rate of T7 polymerase under the influence of activator ROSoxyR complex, promoter trxC and terminators downstream; <html><span lang="latex">\PoPST7\times{T7 effect}\times\frac{N}{V}</span></html>, similarly, represents the synthesizing rate of T7polymerase under the influence of T7promoter; <html><span lang="latex">\-{KdegT7}\times{T7}</span></html>represents the natural degrading rate of T7 polymerase.<br>
+[[File:NYMU_ethanol control .jpg]]
-<html>
+Figure2:This picture is a control group model. It shows that without ''Nosema'' infection (ROS concentration=0), ethanol production is low enough to be ignored. As a result, the bee will not be harmed by ethanol.
-<div lang="latex" class="equation">
-\frac{d[mRNAPDC]}{dt}=\frac{ [T7]^{nT7} }{ KdT7^{nT7}+[T7]^{nY7} }\times{PoPST7}\times\frac{N}{V}-KdegmRNA\times[mRNAPDC]
-</div>
-</html>
-<html>
+[[File:NYMU_ethanol turn off1 .jpg]]
-<div lang="latex" class="equation">
-\frac{d[mRNAADH]}{dt}=\frac{ [T7]^{nT7} }{ KdT7^{nT7}+[T7]^{nY7} }\times{PoPST7}\times\frac{N}{V}-KdegmRNA\times[mRNAADH]
-</div>
-</html>
-In these two equations, the production rate of mRNAPDC and mRNAADH is composed of both synthesizing rate and degrading rate. <html><span lang="latex">\frac{ [T7]^{nT7} }{ KdT7^{nT7}+[T7]^{nY7} }\times{PoPST7｝\times\frac{N}{V}</span></html> represents how T7 polymerase influences promoter T7 on synthesizing mRNAPDC and mRNAADH;<html><span lang="latex">\frac{ [T7]^{nT7} }{ KdT7^{nT7}+[T7]^{nY7} }</span></html> and<html><span lang="latex">\frac{ [T7]^{nT7} }{ KdT7^{nT7}+[T7]^{nY7} }</span></html> represents the natural degrading rate of mRNAPDC and [mRNAADH].
+Figure3: ethanol production turning off after ''Nosema'' is killed  (step 1)
+[[File:NYMU_ethanol turn off2 .jpg]]
+Figure4: ethanol production turning off after ''Nosema'' is killed  (step 2)
+Figure3, Figure4: The two pictures above show that if ''Nosema'' is killed by kill protein effectively, the ethanol production will soon be turned off (will not reach the effective concentration and decrease to a neglectable low level).
+==Discussion==
-<html>
+Figures show that after sensor AhpCp is activated, ethanol reaches its effective concentration 72-80 hours after the infection, which is the time in ''Nosema'' lifecycle when bees carrying ''Nosema'' spores become incurable and infectious to other bees), In other words, this means if killing protein targeting at ''Nosema'' wasn't produced or functioning properly (click here to see the previous model for killing protein), Beecoli turns against the sick bee to kill it within 3-4days.
-<div lang="latex" class="equation">
-\frac{d[PDC]}{dt}=RBS\times{PoPST7effect}\times\frac{N}{V}-{kdegPDC}\times[PDC}]
-</div>
-</html>
+To make ethanol production on time, we adjusted the expression level of OxyR [[(click here for more information)]], the terminators in front of the desired gene, and the promoter strength for independent production of PDC and ADH[[(click here for detailed information about circuit design)]].
-<html>
+==Circuit Design and Improvements==
-<div lang="latex" class="equation">
-\frac{d[ADH]}{dt}=RBS\times{PoPST7effect}\times\frac{N}{V}-{KdegADH}\times[ADH]}
-</div>
-</html>
-<br>
-   In these two equations, the production rate of PDC and ADH is composed of both synthesizing rate and degrading rate. RBS represents ribosome binding site strength, which is the affinity of ribosome to the starting site of mRNA; PoPST7 represents the promoter strength of T7 promoter.<br>
-   For the section of the equation, RBS ×PoPS"T7effect ×N/V"  represents the synthesizing rate of PDC and ADH under the influence of T7promoter; "- KdegPDC ×[PDC]"  and" - KdegADH ×[ADH]"  represents the natural degrading rate of PDC and ADH.<br>
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-\frac{d[ethanol]}{dt}=Kpyruvateacetaldehyde\times{Kacetaldehydeethanol}\times\frac{[PDC]^{nPDC} }{ KdPDC^{nPDC}+[PDC]^{nPDC}}\times\frac{[ADH]^{nADH} }{ KdADH^{nADH}+[ADH]^{nADH}}-Km\times\frac{[ADH]^{nADH}}{ KdADH^{nADH}+[ADH]^{nADH}}\times[ethonal]
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-   In this equation, the production rate of ethanol is composed of both synthesizing rate and degrading rate. Kpyruvateacetaldehyde represents reaction rate constant of pyruvate→acetaldehyde, while Kacetaldehydeethanol  represents reaction rate constant of acetaldehyde→ethanol; 〖[PDC]〗^nPDC/(〖KdPDC〗^nPDC+[PDC]) represents hill effect of PDC to pyruvate→acetaldehyde reaction, while 〖[ADH]〗^nADH/(〖KdADH〗^nADH+[ADH])represents hill effect of ADH to acetaldehyde  ethanol reaction; Km represents the rate constant of ethanol→acetaldehyde, a reverse reaction of the second process of ethanol synthesis.
-For the section of the equation, Kpyruvateacetaldehyde×Kacetaldehydeethanol×  〖[PDC]〗^nPDC/(〖KdPDC〗^nPDC+[PDC])  ×〖[ADH]〗^nADH/(〖KdADH〗^nADH+[ADH]) represents the synthesizing rate of ethanol from pyruvate to acetaldehyde to ethanol under the influence of enzymes ADH and PDC; -Km×[ADH]^nADH/(〖KdADH〗^nADH+[ADH]^nADH )  ×[ethanol] takes the reverse reaction of acetaldehyde to ethanol into consideration, which results in the decreasing of ethanol.
+To postpone ethanol production until it is needed, we adjusted the terminators’ effect in terms of possibility (click here to see the ODE). Results show that there’s a short lag (8-10 hours) between invasion sensing and ethanol production rather than a long one expected. This means that possibility isn’t a sensitive parameter and therefore adding terminator is not an efficient way to manipulate timing when ethanol is produced. Nevertheless, these terminators still has the function of preventing leakage of T7 polymerase production [[(click here for detailed information about circuit design)]].
+By adjusting the parameters in the ODEs given, we found that the nature of T7 transcription is key to adjusting ethanol production time. If postponing ethanol production is needed, a T7 promoter with lower promoter strength and T7 polymerase with higher degrading rate should be chosen
-==parameters:==
+==Parameters==
 {| class="wikitable"
 !Model!!Parameter!!Description!!Value!!Unit!!Reference