NTU@iGEM Project Introduction Methodology Team Introduction Team background Notebook Dry Lab Introduction ODEs used Modeling Constructs Parameters used Deterministic Modeling Sensitivity Analysis Stochastic Modeling Wetlab Introduction Materials and Equipment Protocols Verification of Detection & Lysis System Our New Parts Characterization Introduction Standard Promoters Characterization of pLacI-GFP pLacI-GFP images and clips Characterization of pLsrA-YFP New characterization Proposal Parameter Estimation & Correlations Acknowledgements

Contents 1 Variables 1.1 Lactose controlled production of E7 + Imm 1.2 Iron and Ai2 controlled production of Lysis 2 Parameter Estimation 2.1 E7 production system 2.2 Lysis production system 2.3 GFP production system with logistic growth 2.4 References

Variables

Lactose controlled production of E7 + Imm

1. LacI = A
2. Lactose =B
3. E7 = C

Iron and Ai2 controlled production of Lysis

1. Ai-2 : A
2. ai-2-phos : B
3. LsrR : C
4. SupD derivatives : D
5. T7ptag : E
6. Lysis : F

Parameter Estimation

Estimation of different parameters

1. Transcription  : 70nt/s
2. Translation : 40aa/s
3. Number of Essential Genes : 297
4. Number of mRNA per cell : 4000
5. Average mRNA half life : 5min
6. Average mRNA length  : 1100

Assumptions

1. Rate of transcription is dependent on length of gene
2. Number of amino acids is 1/3 of the number of nucleotides in a gene
3. Rate of Translation is dependent on number of nucleotides
4. For each gene mRNA = 10 at steady state
5. Rate of degradation of average mRNA = 1100/ 5 min
6. Rate of degradation of protein is equivalent to time for cell division i.e. 40 min

E7 production system

Type	Parameter	Values	Comments
Transcription Rate of Lac I gene	k1A	21	Made using Earlier assumptions
Transcription Rate of E7 + Imm gene	k1C	2.470588	Made using Earlier assumptions
Degradation Rate of Lac I mRNA	d1A	0.76246	Made using Earlier assumptions
Transcription Rate of E7 + Imm mRNA	d1C	0.0897	Made using Earlier assumptions
Translation Rate of Lac I mRNA	k2A	36	Made using Earlier assumptions
Translation Rate of E7 + Imm mRNA	k2C	4.23539	Made using Earlier assumptions
Protein Degradation Rate	d2A,d2C	0.03465	Made using Earlier assumptions
Hill coefficeint for E7 + Imm	nC	1	This is obtained on the assumption that one Repressor Protein binds to one Lactose molecule complex
Dissociation Constant for E7 + Imm	KC	0.8	[1]
Constitutive Portion for E7 + Imm	aC	0.5	Estimate since a is between 0 and 1 Implication that Lactose may not be a very strongly Regulated Promoter
Complex Formation Rate Between Lac I Repressor and Lactose	k3AB	1	Estimate. This is based on the assumption that the complex formation is only dependent on the concentrations of Lac I repressor and Lactose

Lysis production system

Some of the parameter values were obtained via trial and error as the values could not be found in literature. The values are chosen to produce the desired behaviour of the graph. More work has to be done to actually find out how much lysis protein quantity is really required for lysis of the cell as this is still an unknown. Readers may find some of the parameters unrealistic but the purpose of this exercise is to observe how changing certain parameters may affect the system overall output. We advise the reader to look more into the behaviour of the system rather than be focused upon the numerical values.

Type	Parameter	Values	Comments
Phosphorylation Rate of Ai-2	kPF	1	Estimate
Dephosphorylation Rate of Ai-2	kPB	1	Estimate
Transcription Rate of LsrR gene	k1C	4.402517	Made using Earlier assumptions
Transcription Rate of SupD gene	k1D	46.667	Made using Earlier assumptions
Transcription Rate of t7pTag gene	k1E	1.5556	Made using Earlier assumptions
Transcription Rate of Lysis gene	k1F	28	Made using Earlier assumptions
Degradation Rate of LsrR mRNA	d1C	0.159845	Made using Earlier assumptions
Degradation Rate of SupD mRNA	d1D	1.694359	Made using Earlier assumptions
Degradation Rate of t7pTag mRNA	d1E	0.056478	Made using Earlier assumptions
Degradation Rate of Lysis mRNA	d1F	1.0166	Made using Earlier assumptions
Translation Rate of LsrR Protein	k2C	7.54716	Made using Earlier assumptions
Translation Rate of t7 pTag Protein	k2E	2.6667	Made using Earlier assumptions
Translation Rate of Lysis Protein	k2F	48	Made using Earlier assumptions
Protein Degradation Rate	d2C,d2E,d2F	0.03465	Made using Earlier assumptions
Hill coefficeint for SupD	nD	50	Trial And Error
Hill coefficeint for t7 pTag	nE	1	Estimate. It is assuming that one molecule of iron ion is required to activate the production of one t7mRNA
Hill coefficeint for Lysis	nF	1	Estimate. It is assumed that one molecule of t7 is required for activation of one Lysis mRNA
Dissociation Constant for SupD	KD	15	[2]
Dissociation Constant for t7 pTag	KE	1	Trial And Error
Dissociation Constant for Lysis	KF	1	Trial And Error
Constitutive Portion for SupD	aD	0.01	Trial and Error
Constitutive Portion for t7 pTag	aE	0.01	Trial And Error
Constitutive Portion for Lysis	aF	0.0001	Trial and Error
Complex Formation Rate Between LsrR Repressor and Ai-2-Phosphorylated	k3BC	0.01	Trial and Error
Complex Formation Rate Between SupD tRNA and t7 pTag mRNA	k3DE	0.000000001	Trial and Error
Amount of Protein Kinase	S	28.747	From Earlier Assumptions

GFP production system with logistic growth

Type	Parameter	Values	Comments
Forward Reaction rate of Complex	kf	1.2	Trial And Error
Reverse Reaction rate of Complex	kr	1.2	Trial And Error

References

1. [http://parts.mit.edu/igem07/index.php?title=ETHZ/Parameters http://parts.mit.edu/igem07/index.php?title=ETHZ/Parameters]

2. Grass G, Otto M, Fricke B, et al., FieF (YiiP) from Escherichia coli mediates decreased cellular accumulation of iron and relieves iron stress, ARCHIVES OF MICROBIOLOGY, Volume: 183 Issue: 1 Pages: 9-18 Published: JAN 2005