Difference between revisions of "Part:BBa K1197013"

Latest revision as of 03:33, 5 October 2013

CYP6G1 - Insecticide resistance gene

It is an insecticide resistance gene which is found in Drosophila melanogaster. We used it in our system to metabolize imidacloprid by producing nontoxic byproducts.

It is obtained from Drosophila melanogaster genome and modified according to work in B.subtilis.

Original sequence was modified since it was not proper for prokaryotic expression. Its first 21 nucleotide which coding 7 aa was changed with "Barnes" sequence: MVLTEVL is the native aa sequence, and MALLLAV is the Barnes aa sequence. (Barnes et al. 1991)

Reference: Barnes, H.J., Arlotto, M.P., Waterman, M.R., 1991. Expression and enzymatic activity of recombinant cytochrome P450 17a-hydroxylase in Escherichia coli. Proc. Natl. Acad. Sci. USA 88, 5597e5601.

• P450 Monooxgenase Production and its Activity:

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.

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.

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 542
Illegal XhoI site found at 1210
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 943

@@ Line 11: / Line 11: @@
 of recombinant cytochrome P450 17a-hydroxylase in ''Escherichia coli''. Proc. Natl.
 Acad. Sci. USA 88, 5597e5601.
-• P450 Monooxgenase Production and its Activity:
-Overview:
-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 :
+• P450 Monooxgenase Production and its Activity:
+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.
-PARAMETERS
+https://static.igem.org/mediawiki/parts/8/87/P450.jpg
-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 42: / Line 24: @@
 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;
-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]
+https://static.igem.org/mediawiki/parts/7/73/4-hydroxy.jpg
-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.
-References:
-- http://www.ncbi.nlm.nih.gov/pubmed/23470655
-- http://www.bioone.org/doi/abs/10.1603/0022-0493-101.6.1743
-- http://www.sciencedirect.com/science/article/pii/S0048357512000685
 <!-- Add more about the biology of this part here
 ===Usage and Biology===