Difference between revisions of "Part:BBa K1197015"

Revision as of 03:56, 5 October 2013

Catalytically active CYP6G1 coding device

This device was constructed to produce catalytically active CYP6G1 in B. subtilis and secrete it outside. Secretion is needed since CYP6G1 enzyme should degrade imidacloprid in bee midgut. To provide constitutively production and secretion of this enzyme, we used Pveg constitutive promoter, SpoVG RBS with SacB signal peptide (K541501) which is used by Fatih_Turkey Team in 2011.

Also fCPR production is necessary to make CYP6G1 enzyme catalytically active. Thus, SpoVG RBS and SacB signal peptide was used before fCPR too.

• P450 Monooxgenase Production and its Activity:

CYP6G1 gene which will produce P450, is synthesized by using B. subtilis specific constituve promoter Pveg. fCPR gene, used for activation of CYP6G1 in B. subtilis, is also transcribed with same promoter. These two genes has transcribed with SacB genes, after translation which helps to export these proteins through outside of the cell, into the bee gut. [1] To be able know, how much enzyme is produced in a given time and how much imidacloprid is converted into nontoxic components by P450, model of this circuit was required.

Enzyme Kinetics for P450 Monooxygenase:

To be able to estimate the rate of the P450 reaction with imidacloprid, we used Michaelis-Menten Kinetics. The harmful imidacloprid concentration for honey bees is reported as 1200 µg/l [2], so in our model we assumed that the imidacloprid concentration in bee gut as 1200 µg/l. And P450 monooxgenase will be produced from our circuit and directly secreted into bee gut where imidacloprid is found.

According to Michaelis-Menten Kinetics;

• So according to these parameters, we have calculated our enzyme activity according to our enzyme production rate. In 3500 s , 4E10 molecule of imidocloprid is degraded according to our model.

References:

1- http://www.ncbi.nlm.nih.gov/pubmed/23470655 2- http://www.bioone.org/doi/abs/10.1603/0022-0493-101.6.1743 3- http://www.sciencedirect.com/science/article/pii/S0048357512000685 Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 767
Illegal BamHI site found at 3178
Illegal XhoI site found at 1435
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1168
Illegal BsaI.rc site found at 3700

@@ Line 8: / Line 8: @@
 • P450 Monooxgenase Production and its Activity:
-Overview:
+CYP6G1 gene which will produce P450, is synthesized by using B. subtilis specific constituve promoter Pveg. fCPR  gene, used for activation of CYP6G1 in B. subtilis, is also transcribed with same promoter. These two genes has transcribed with SacB genes, after translation which helps to export these proteins through outside of the cell, into the bee gut. [1]  To be able know, how much enzyme is produced in a given time and how much imidacloprid is converted into nontoxic components by P450,  model of this circuit was required.
-The aim of modeling P450 monooxygenase production is to predict the amount and the activity of P450 enzyme which will be produced from our circuit . The model was created mainly by designing ODEs and enzyme kinetics equations .Then; these equations were solved by the help of MATLAB software.
-As seen in the circuit, CYP6G1 gene , which will produce P450, is synthesized by using B. subtilis specific constituve promoter Pveg. fCPR  gene, used for activation of CYP6G1 in B. subtilis, is also transcribed with same promoter. These two genes has transcribed with SacB genes, after translation which helps to export these proteins through outside of the cell, into the bee gut. [1]  To be able know, how much enzyme is produced in a given time and how much imidacloprid is converted into nontoxic components by P450,  model of this circuit was required.
-Mathematical Model :
-It is always the best creating flowcharts as modeling systems. And also our modeling team is an interdisciplinary, it makes easier to be understood by all of us.
-Differential Equations :
-PARAMETERS
-N             Plasmid Copy Number
-P             Promoter Strength
-amrna     Degradation rate of mRNA
-K             Dissociation Constant
-N             Hill Coefficient
-T             Translation Rate
-D             Protein Degradation Rate
-Simulations:
 Enzyme Kinetics for P450 Monooxygenase:
@@ Line 67: / Line 16: @@
   According to Michaelis-Menten Kinetics;
-We applied this equation as:
-While we were looking for Km and Kcat parameters of P450(from CYP6G1 gene), we couldn’t find  them for A. mellifera but we used parameter of another gene producing P450, which is CYP6CM1 vQ. From homology modeling , it has the closest relation with our P450 , produced from CYP6G1. [3]
-from aminoacid sequence:
-from homology modelling:
 • So according to these parameters, we have calculated our enzyme activity according to our enzyme production rate. In 3500 s ,  4E10 molecule of imidocloprid is degraded according to our model.