http://2012.igem.org/wiki/index.php?title=Special:Contributions/RoMann&feed=atom&limit=50&target=RoMann&year=&month=2012.igem.org - User contributions [en]2024-03-29T12:27:58ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:Tuebingen/SafetyTeam:Tuebingen/Safety2012-09-04T13:30:03Z<p>RoMann: /* Safety */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
<br />
== Safety ==<br />
<br />
''Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?''<br />
<br />
All our work is supervised by PhD students, PhDs and professors of our university who are very experienced in lab work and are currently working in genetics. We document our work in our lab notebook to enable replicability and prevent unnecessary errors.<br />
<br />
Our project does not pose any dangers to researchers or the public: We are following widely used lab protocols in a appropriate lab facility. The organisms used in our project, namely ''E. coli'' (TOP10 strain) and ''S. cerevisiae'', are well-known and considered safe.<br />
<br />
Although, by building a biosensor we are tackling an environmental problem, our system is not intended to be used in the field but in a lab only.<br />
<br />
<br />
''Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?''<br />
<br />
We have designed 3 different types of BioBricks. Membrane bound receptors for sensing hormones, a small inverter system to regulate our reporter gene. None of these are found toxic or dangerous by any means.<br />
The receptors we use are synthesized after genome-sequences of ''Danio rerio'' and ''Xenopus laevis'', both are commonly used model organisms. The rest of the genes and promoters are extracted from the ''Saccharomyces cerevisiae'' genome via PCR. All of the parts we use are expressed in wildtype organisms and not known to have any harmful effect on humans or other organisms.<br />
<br />
<br />
''Which specific biosafety rules or guidelines do you have to consider in your country?''<br />
<br />
In Germany any work with genetically modified organisms is regulated by the "Gentechnikgesetz". There are different biosafety levels reaching from 1 to 4. Our lab has been registered as level 1.<br />
Our work matches the definition of level 1 since on our current understanding we see no threats on human health or the environment.<br />
<br />
Genetically modified organisms are allowed to be released into nature only on permission of the Umweltministerium and once freed need to be constantly monitored.<br />
<br />
<br />
''Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?''<br />
<br />
Parts that are able to induce apoptosis or autolyis could be expressed under promoters that are activated if a specific substance is lacking or present in an uncontrolled environment as opposed to a lab. As an example the team of UCL London had a concept for autolysis that could be of value in [https://2011.igem.org/Team:UCL_London/Research/Extractery/Theory 2011]. A possible promoter could be induced by the lack of iron, which then would have to be a part of the culture medium in the lab. In environments without iron the organism not only couldn't survive but would be killed immediately. A machanism of this kind would then have to be a obligatory requirement for all contest-submissions.</div>RoMannhttp://2012.igem.org/Team:Tuebingen/ProjectTeam:Tuebingen/Project2012-09-04T13:06:46Z<p>RoMann: /* Occurring Questions */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
<br />
__TOC__<br />
<br />
== Overview ==<br />
<br />
Our general aim is to establish a simple synthetic organisim which will<br />
be capable to measure the influence of endocrine disruptors on the<br />
natural balance of sexual determination in all kind of vertebrates. The<br />
measurement itself will be cost-efficient, environment-friendly and<br />
sensitive.<br />
<br />
Naturally occuring iron receptors of the PAQR family are found to<br />
repress the fet3 promotor on high level of extra-cellular iron.<br />
According to Smith et al. human mPR expressed in yeast induced the same<br />
signal when binding to progesterone. Relying on this results we will<br />
express various mPRs of Danio rerio and Xenopus laevis in yeast to<br />
measure endocrine substances that influence fish and amphibians.<br />
<br />
We will transform the negative signal (fet3 repression) into a positive<br />
signal by regulating a repressor (rox1 or mig1) with Pfet3. This<br />
repressor will in turn regulate the expression of the reporter gene<br />
(firefly luciferase or beta-galactosidase) and allow quantitative<br />
measurement.<br />
<br />
== Motivation ==<br />
<br />
''Why do we want to establish a mechanism for steroid measurement?''<br />
<br />
Steroid hormones, especially estrogens, occur in all vertebrates and<br />
play a crucial role in sexual differentiation. In recent times the<br />
pollution of waters with these hormones has become an increasing problem<br />
for the aquatic fauna.<br />
<br />
Particularly waters functionalized by humans or adjacent to human<br />
settlements, e.g. in areas with agricultural use, show increased<br />
concentrations of estrogen.<br />
<br />
Scientific studies based on ''Danio rerio'' showed that the consequences<br />
are devastating.<br />
<br />
High concentrations of 17α-ethinylestradiol, a hormone in most<br />
birth-control pills, affected the sex differentiation of ''Danio rerio''<br />
leading to development of ovotestis or complete feminization (Andersenc,<br />
2002).<br />
<br />
Intersex-fish have been reported in UK rivers since 1978 downstream of<br />
an sewage treatment plant.<br />
<br />
We believe that a first step in finding a solution to this environmental<br />
problem is an accurate and reliable method to quantify steroid<br />
concentrations.<br />
<br />
== Occurring Questions ==<br />
<br />
On our way designing the major pathway to express a specific reporter<br />
gene to demonstrate the presence of steroid hormones, we had and still<br />
have to deal with several questions concering the choice of BioBricks,<br />
genes and vectors to construct a firm method to determine "pollution" by<br />
steroids. As a conclusion, we have to meet two major requirements for<br />
our system:<br />
<br />
* It should be as cost-efficient as possible for easy and regular application<br />
* It should be resistant to yeast's own metabolism (Not be disturbed by unexpected occuring expression).<br />
<br />
At first we had to find an appropriate receptor to "grab" steroid<br />
hormones in efluents. This should fulfill the following requirements:<br />
<br />
* It should only be responsible to substrates we wish to detect, so the results of the test will not be falsified.<br />
* It has to be easiliy integrated into yeast's cell membrane.<br />
* Its nucleic acid sequence should not be too long so we can put it onto a plasmid vector.<br />
<br />
We chose the membrane progesterone receptor of the zebra fish (''Danio rerio'') and the African clawed frog (''Xenopus laevis''). We focused on<br />
these receptors, since they are easy to duplicate and interact with a<br />
broad bandwith of sex-determining hormones.<br />
<br />
The next step was to select an appropriate organism to express these<br />
receptors. After some research, we could narrow our options down to two<br />
organisms:<br />
<br />
* ''Escherichia coli''<br />
* ''Saccharomyces cerevisiae''<br />
<br />
Finally we decided for yeast, since it has been done more research with<br />
it according to our prefered receptors. In addition yeast is an<br />
eukaryote making it more easier to integrate mPRs into their cell<br />
membrane.<br />
<br />
== '''Mechanism''' ==<br />
<br />
[[File:Igem_2.3.jpg|thumb|right|Planned mechanism]]<br />
<br />
Naturally occuring iron receptors of the PAQR family are found to block<br />
FET3 promotor on high level of extra cellular iron. According to<br />
[http://www.ncbi.nlm.nih.gov/pubmed/18603275 Smith JL et al] expressed<br />
human mPR in yeast induced the same signal ligating to estrogen. Relying<br />
on this results we will try to express various mPRs of ''Danio rerio'',<br />
''Xanophus laevis'' in yeast which we find better fitting to measure<br />
endocrine substances that influence fish.<br />
<br />
We will transform the negative signal of FET3 by letting it control an<br />
inhibitor (ROX1 and MIG1). This inhibitor will regulate the promotor of<br />
our reporter gene. For the promoter of our reporter gene we chose ANB1<br />
and SUC2, because these are targets of ROX1 and MIG1. By negating the<br />
negative signal of FET3 we hope to obtain a positive signal sensitive<br />
enough to measure low concentrations of different endocrine substances.<br />
<br />
=== Receptors ===<br />
<br />
Membrane bound, 7-Transmembranereceptor (C-terminus inside,N-terminus<br />
outside), PAQR family (progesterone adiponectin Q receptor), Hly-III<br />
superfamily G-Protein coupled: activation of inhibitori Gi units:<br />
reduced adenylyl-cyclase activity. <br />
<br />
=== Inhibitors and their targets ===<br />
<br />
An appropriate inhibitor/promotor combination is a crucial step in our<br />
pathway and should be selected wisely.<br />
<br />
Finally we chose FET3 as our promoter and both ROX1 and MIG1 as<br />
inhibitors. It is very likely, that they don't seem to repress any gene<br />
expression that are crucial for yeast.<br />
<br />
=== Reporter genes ===<br />
<br />
The last station of our signaling pathway should be a reporter gene<br />
which amplifies our initial signal to allow a quantitative measurement.<br />
<br />
The enzyme '''luciferase''' fulfills these conditions and is our<br />
candidate of choice.<br />
<br />
== Implementation ==<br />
<br />
<br />
=== Promoter ===<br />
[[File:PRS313_Padh1_Danio_Tadh1.png|promoter1]]<br />
[[File:PRS313_Padh1_Xenopus_Tadh1.png|promoter2]]<br />
=== Inhibitor ===<br />
[[File:PRS315_Pfet3_rox1_Tadh1_V3.png|inhibitor1]]<br />
[[File:pRS315_Pfet3_mig1_Tadh1.png|inhibitor2]]<br />
=== Reporter ===<br />
[[File:PRS316_Panb1_lacZ_Tadh1.png|reporter1]]<br />
[[File:pRS316_Psuc2_lacZ_Tadh1.png|reporter2]]<br />
[[File:PRS316_Psuc2_Luciferase_Tadh1.png|reporter3]]<br />
<br />
== Measurement ==<br />
<br />
The measurement itself should be limited to an optical one. With this<br />
idea in mind, we decided for reporter genes like lacZ and luciferase,<br />
because these produce signals which can simply be quantified by optical<br />
measurement methods.<br />
<br />
== References ==<br />
<br />
* [1] Jessica L. Smith, Brian R. Kupchak, Ibon Garitaonandia, L. Kim Hoang, Andrew S. Maina, Lisa M. Regalla, and Thomas J. Lyons - 2008 - Heterologous expression of human mPRα, mPRβ and mPRγ in yeast confirms their ability to function as membrane progesterone receptors<br />
* [2] Sumpter, Johnson (2008) Reflections on endocrine disruption in the aquatic environment<br />
<!--<br />
* Liew et. al. - 2012 Polygenic Sex Determination System in Zebrafish<br />
* Hanna et al. - 2006 - Cell-surface expression, progestin binding, and<br />
rapid nongenomic signaling of zebrafish membrane progestin receptors<br />
alpha and beta in transfected cells<br />
* Reupke - 2011 - Detektion dopingrelevanter anaboler Steroide in<br />
Pferdeurin und Pferdeplasma mithilfe eines Reportergen-Assays in<br />
Hefezellen. (Detection of doping relevant anabolic steroids in horse<br />
urin and blood plasma using yeast reporter gene assays)<br />
* “Assault on the male”, BBC 1996. Video report<br />
http://www.youtube.com/watch?v=LkxIJJI37bQ<br />
* Dr. Volker Scheil, The impact of potential environmental stressors on<br />
early development and cellular and biochemical biomarkers in fish,<br />
Main research: Fish embryotoxicity, histopathology and stress protein<br />
(hsp 70) responses.<br />
* Harris et al. - 2011 - The consequences of feminization in breeding<br />
groups of wild fish<br />
* Liew et al. - 2012 - Polygenic Sex Determination System in Zebrafish<br />
* Sumpter, Johnson - 2008 - 10th Anniversary Perspective Reflections on<br />
endocrine disruption in the aquatic environment from known knowns to<br />
unknown unknowns (and many things in between)<br />
* Werner, Palace, Wautier - 2006 - Reproductive fitness of lake trout<br />
(Salvelinus namaycush) exposed to environmentally relevant<br />
concentrations of the potent estrogen ethynylestradiol (EE2) in a<br />
whole lake exposure experiment<br />
* Fenske, M., Maack, G., Schäfers, C., & Segner, H. (2005). An<br />
environmentally relevant concentration of estrogen induces arrest of<br />
male gonad development in zebrafish, Danio rerio. Environmental<br />
toxicology and chemistry / SETAC, 24(5), 1088-98. Retrieved from<br />
http://www.ncbi.nlm.nih.gov/pubmed/16110986 (kein PDF)<br />
* Schäfers, C., Teigeler, M., Wenzel, A., Maack, G., Fenske, M., &<br />
Segner, H. (2007). Concentration- and time-dependent effects of the<br />
synthetic estrogen, 17alpha-ethinylestradiol, on reproductive<br />
capabilities of the zebrafish, Danio rerio. Journal of toxicology and<br />
environmental health. Part A, 70(9), 768-79.<br />
doi:10.1080/15287390701236470<br />
* Segner, H., Caroll, K., Fenske, M., Janssen, C. R., Maack, G., Pascoe,<br />
D., Schäfers, C., et al. (2003). Identification of<br />
endocrine-disrupting effects in aquatic vertebrates and invertebrates:<br />
report from the European IDEA project. Ecotoxicology and environmental<br />
safety, 54(3), 302-14. Retrieved from<br />
http://www.ncbi.nlm.nih.gov/pubmed/12651186 (kein PDF)<br />
* Hanna et al. - 2006 - Cell-surface expression, progestin binding, and<br />
rapid nongenomic signaling of zebrafish membrane progestin receptors<br />
alpha and beta in transfected cells<br />
* Thomas et al. - 2007 - Steroid and G protein binding characteristics<br />
of the seatrout and human progestin membrane receptor alpha subtypes<br />
and their evolutionary origins<br />
* Thomas - 2008 - Characteristics of membrane progestin receptor alpha<br />
(mPR ) and progesterone membrane receptor component 1 (PGMRC1) and<br />
their roles in mediating rapid progestin actions<br />
* Jessica L. Smith, Brian R. Kupchak, Ibon Garitaonandia, L. Kim Hoang,<br />
Andrew S. Maina, Lisa M. Regalla, and Thomas J. *Lyons - 2008 -<br />
Heterologous expression of human mPRα, mPRβ and mPRγ in yeast confirms<br />
their ability to function as membrane progesterone receptors<br />
<br />
--></div>RoMannhttp://2012.igem.org/Team:Tuebingen/ProjectTeam:Tuebingen/Project2012-09-04T13:00:45Z<p>RoMann: /* Occurring Questions */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
<br />
__TOC__<br />
<br />
== Overview ==<br />
<br />
Our general aim is to establish a simple synthetic organisim which will<br />
be capable to measure the influence of endocrine disruptors on the<br />
natural balance of sexual determination in all kind of vertebrates. The<br />
measurement itself will be cost-efficient, environment-friendly and<br />
sensitive.<br />
<br />
Naturally occuring iron receptors of the PAQR family are found to<br />
repress the fet3 promotor on high level of extra-cellular iron.<br />
According to Smith et al. human mPR expressed in yeast induced the same<br />
signal when binding to progesterone. Relying on this results we will<br />
express various mPRs of Danio rerio and Xenopus laevis in yeast to<br />
measure endocrine substances that influence fish and amphibians.<br />
<br />
We will transform the negative signal (fet3 repression) into a positive<br />
signal by regulating a repressor (rox1 or mig1) with Pfet3. This<br />
repressor will in turn regulate the expression of the reporter gene<br />
(firefly luciferase or beta-galactosidase) and allow quantitative<br />
measurement.<br />
<br />
== Motivation ==<br />
<br />
''Why do we want to establish a mechanism for steroid measurement?''<br />
<br />
Steroid hormones, especially estrogens, occur in all vertebrates and<br />
play a crucial role in sexual differentiation. In recent times the<br />
pollution of waters with these hormones has become an increasing problem<br />
for the aquatic fauna.<br />
<br />
Particularly waters functionalized by humans or adjacent to human<br />
settlements, e.g. in areas with agricultural use, show increased<br />
concentrations of estrogen.<br />
<br />
Scientific studies based on ''Danio rerio'' showed that the consequences<br />
are devastating.<br />
<br />
High concentrations of 17α-ethinylestradiol, a hormone in most<br />
birth-control pills, affected the sex differentiation of ''Danio rerio''<br />
leading to development of ovotestis or complete feminization (Andersenc,<br />
2002).<br />
<br />
Intersex-fish have been reported in UK rivers since 1978 downstream of<br />
an sewage treatment plant.<br />
<br />
We believe that a first step in finding a solution to this environmental<br />
problem is an accurate and reliable method to quantify steroid<br />
concentrations.<br />
<br />
== Occurring Questions ==<br />
<br />
On our way designing the major pathway to express a specific reporter<br />
gene to demonstrate the presence of steroid hormones, we had and still<br />
have to deal with several questions concering the choice of BioBricks,<br />
genes and vectors to construct a firm method to determine "pollution" by<br />
steroids. As a conclusion, we have to meet two major requirements for<br />
our system:<br />
<br />
* It should be as cost-efficient as possible for easy and regular<br />
application<br />
* It should be resistant to yeast's own metabolism (Not be disturbed by<br />
unexpected occuring expression).<br />
<br />
At first we had to find an appropriate receptor to "grab" steroid<br />
hormones in efluents. This should fulfill the following requirements:<br />
<br />
* It should only be responsible to substrates we wish to detect, so the<br />
results of the test will not be falsified.<br />
* It has to be easiliy integrated into yeast's cell membrane.<br />
* Its nucleic acid sequence should not be too long so we can put it onto<br />
a plasmid vector.<br />
<br />
We chose the membrane progesterone receptor of the zebra fish (''Danio<br />
rerio'') and the African clawed frog (''Xenopus laevis''). We focused on<br />
these receptors, since they are easy to duplicate and interact with a<br />
broad bandwith of sex-determining hormones.<br />
<br />
The next step was to select an appropriate organism to express these<br />
receptors. After some research, we could narrow our options down to two<br />
organisms:<br />
<br />
* ''Escherichia coli''<br />
* ''Saccharomyces cerevisiae''<br />
<br />
Finally we decided for yeast, since it has been done more research with<br />
it according to our prefered receptors. In addition yeast is an<br />
eukaryote making it more easier to integrate mPRs into their cell<br />
membrane.<br />
<br />
== '''Mechanism''' ==<br />
<br />
[[File:Igem_2.3.jpg|thumb|right|Planned mechanism]]<br />
<br />
Naturally occuring iron receptors of the PAQR family are found to block<br />
FET3 promotor on high level of extra cellular iron. According to<br />
[http://www.ncbi.nlm.nih.gov/pubmed/18603275 Smith JL et al] expressed<br />
human mPR in yeast induced the same signal ligating to estrogen. Relying<br />
on this results we will try to express various mPRs of ''Danio rerio'',<br />
''Xanophus laevis'' in yeast which we find better fitting to measure<br />
endocrine substances that influence fish.<br />
<br />
We will transform the negative signal of FET3 by letting it control an<br />
inhibitor (ROX1 and MIG1). This inhibitor will regulate the promotor of<br />
our reporter gene. For the promoter of our reporter gene we chose ANB1<br />
and SUC2, because these are targets of ROX1 and MIG1. By negating the<br />
negative signal of FET3 we hope to obtain a positive signal sensitive<br />
enough to measure low concentrations of different endocrine substances.<br />
<br />
=== Receptors ===<br />
<br />
Membrane bound, 7-Transmembranereceptor (C-terminus inside,N-terminus<br />
outside), PAQR family (progesterone adiponectin Q receptor), Hly-III<br />
superfamily G-Protein coupled: activation of inhibitori Gi units:<br />
reduced adenylyl-cyclase activity. <br />
<br />
=== Inhibitors and their targets ===<br />
<br />
An appropriate inhibitor/promotor combination is a crucial step in our<br />
pathway and should be selected wisely.<br />
<br />
Finally we chose FET3 as our promoter and both ROX1 and MIG1 as<br />
inhibitors. It is very likely, that they don't seem to repress any gene<br />
expression that are crucial for yeast.<br />
<br />
=== Reporter genes ===<br />
<br />
The last station of our signaling pathway should be a reporter gene<br />
which amplifies our initial signal to allow a quantitative measurement.<br />
<br />
The enzyme '''luciferase''' fulfills these conditions and is our<br />
candidate of choice.<br />
<br />
== Implementation ==<br />
<br />
<br />
=== Promoter ===<br />
[[File:PRS313_Padh1_Danio_Tadh1.png|promoter1]]<br />
[[File:PRS313_Padh1_Xenopus_Tadh1.png|promoter2]]<br />
=== Inhibitor ===<br />
[[File:PRS315_Pfet3_rox1_Tadh1_V3.png|inhibitor1]]<br />
[[File:pRS315_Pfet3_mig1_Tadh1.png|inhibitor2]]<br />
=== Reporter ===<br />
[[File:PRS316_Panb1_lacZ_Tadh1.png|reporter1]]<br />
[[File:pRS316_Psuc2_lacZ_Tadh1.png|reporter2]]<br />
[[File:PRS316_Psuc2_Luciferase_Tadh1.png|reporter3]]<br />
<br />
== Measurement ==<br />
<br />
The measurement itself should be limited to an optical one. With this<br />
idea in mind, we decided for reporter genes like lacZ and luciferase,<br />
because these produce signals which can simply be quantified by optical<br />
measurement methods.<br />
<br />
== References ==<br />
<br />
* [1] Jessica L. Smith, Brian R. Kupchak, Ibon Garitaonandia, L. Kim Hoang, Andrew S. Maina, Lisa M. Regalla, and Thomas J. Lyons - 2008 - Heterologous expression of human mPRα, mPRβ and mPRγ in yeast confirms their ability to function as membrane progesterone receptors<br />
* [2] Sumpter, Johnson (2008) Reflections on endocrine disruption in the aquatic environment<br />
<!--<br />
* Liew et. al. - 2012 Polygenic Sex Determination System in Zebrafish<br />
* Hanna et al. - 2006 - Cell-surface expression, progestin binding, and<br />
rapid nongenomic signaling of zebrafish membrane progestin receptors<br />
alpha and beta in transfected cells<br />
* Reupke - 2011 - Detektion dopingrelevanter anaboler Steroide in<br />
Pferdeurin und Pferdeplasma mithilfe eines Reportergen-Assays in<br />
Hefezellen. (Detection of doping relevant anabolic steroids in horse<br />
urin and blood plasma using yeast reporter gene assays)<br />
* “Assault on the male”, BBC 1996. Video report<br />
http://www.youtube.com/watch?v=LkxIJJI37bQ<br />
* Dr. Volker Scheil, The impact of potential environmental stressors on<br />
early development and cellular and biochemical biomarkers in fish,<br />
Main research: Fish embryotoxicity, histopathology and stress protein<br />
(hsp 70) responses.<br />
* Harris et al. - 2011 - The consequences of feminization in breeding<br />
groups of wild fish<br />
* Liew et al. - 2012 - Polygenic Sex Determination System in Zebrafish<br />
* Sumpter, Johnson - 2008 - 10th Anniversary Perspective Reflections on<br />
endocrine disruption in the aquatic environment from known knowns to<br />
unknown unknowns (and many things in between)<br />
* Werner, Palace, Wautier - 2006 - Reproductive fitness of lake trout<br />
(Salvelinus namaycush) exposed to environmentally relevant<br />
concentrations of the potent estrogen ethynylestradiol (EE2) in a<br />
whole lake exposure experiment<br />
* Fenske, M., Maack, G., Schäfers, C., & Segner, H. (2005). An<br />
environmentally relevant concentration of estrogen induces arrest of<br />
male gonad development in zebrafish, Danio rerio. Environmental<br />
toxicology and chemistry / SETAC, 24(5), 1088-98. Retrieved from<br />
http://www.ncbi.nlm.nih.gov/pubmed/16110986 (kein PDF)<br />
* Schäfers, C., Teigeler, M., Wenzel, A., Maack, G., Fenske, M., &<br />
Segner, H. (2007). Concentration- and time-dependent effects of the<br />
synthetic estrogen, 17alpha-ethinylestradiol, on reproductive<br />
capabilities of the zebrafish, Danio rerio. Journal of toxicology and<br />
environmental health. Part A, 70(9), 768-79.<br />
doi:10.1080/15287390701236470<br />
* Segner, H., Caroll, K., Fenske, M., Janssen, C. R., Maack, G., Pascoe,<br />
D., Schäfers, C., et al. (2003). Identification of<br />
endocrine-disrupting effects in aquatic vertebrates and invertebrates:<br />
report from the European IDEA project. Ecotoxicology and environmental<br />
safety, 54(3), 302-14. Retrieved from<br />
http://www.ncbi.nlm.nih.gov/pubmed/12651186 (kein PDF)<br />
* Hanna et al. - 2006 - Cell-surface expression, progestin binding, and<br />
rapid nongenomic signaling of zebrafish membrane progestin receptors<br />
alpha and beta in transfected cells<br />
* Thomas et al. - 2007 - Steroid and G protein binding characteristics<br />
of the seatrout and human progestin membrane receptor alpha subtypes<br />
and their evolutionary origins<br />
* Thomas - 2008 - Characteristics of membrane progestin receptor alpha<br />
(mPR ) and progesterone membrane receptor component 1 (PGMRC1) and<br />
their roles in mediating rapid progestin actions<br />
* Jessica L. Smith, Brian R. Kupchak, Ibon Garitaonandia, L. Kim Hoang,<br />
Andrew S. Maina, Lisa M. Regalla, and Thomas J. *Lyons - 2008 -<br />
Heterologous expression of human mPRα, mPRβ and mPRγ in yeast confirms<br />
their ability to function as membrane progesterone receptors<br />
<br />
--></div>RoMannhttp://2012.igem.org/Team:Tuebingen/ProjectTeam:Tuebingen/Project2012-09-04T12:59:29Z<p>RoMann: /* Occurring Questions */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
<br />
__TOC__<br />
<br />
== Overview ==<br />
<br />
Our general aim is to establish a simple synthetic organisim which will<br />
be capable to measure the influence of endocrine disruptors on the<br />
natural balance of sexual determination in all kind of vertebrates. The<br />
measurement itself will be cost-efficient, environment-friendly and<br />
sensitive.<br />
<br />
Naturally occuring iron receptors of the PAQR family are found to<br />
repress the fet3 promotor on high level of extra-cellular iron.<br />
According to Smith et al. human mPR expressed in yeast induced the same<br />
signal when binding to progesterone. Relying on this results we will<br />
express various mPRs of Danio rerio and Xenopus laevis in yeast to<br />
measure endocrine substances that influence fish and amphibians.<br />
<br />
We will transform the negative signal (fet3 repression) into a positive<br />
signal by regulating a repressor (rox1 or mig1) with Pfet3. This<br />
repressor will in turn regulate the expression of the reporter gene<br />
(firefly luciferase or beta-galactosidase) and allow quantitative<br />
measurement.<br />
<br />
== Motivation ==<br />
<br />
''Why do we want to establish a mechanism for steroid measurement?''<br />
<br />
Steroid hormones, especially estrogens, occur in all vertebrates and<br />
play a crucial role in sexual differentiation. In recent times the<br />
pollution of waters with these hormones has become an increasing problem<br />
for the aquatic fauna.<br />
<br />
Particularly waters functionalized by humans or adjacent to human<br />
settlements, e.g. in areas with agricultural use, show increased<br />
concentrations of estrogen.<br />
<br />
Scientific studies based on ''Danio rerio'' showed that the consequences<br />
are devastating.<br />
<br />
High concentrations of 17α-ethinylestradiol, a hormone in most<br />
birth-control pills, affected the sex differentiation of ''Danio rerio''<br />
leading to development of ovotestis or complete feminization (Andersenc,<br />
2002).<br />
<br />
Intersex-fish have been reported in UK rivers since 1978 downstream of<br />
an sewage treatment plant.<br />
<br />
We believe that a first step in finding a solution to this environmental<br />
problem is an accurate and reliable method to quantify steroid<br />
concentrations.<br />
<br />
== Occurring Questions ==<br />
<br />
On our way designing the major pathway to express a specific reporter<br />
gene to demonstrate the presence of steroid hormones, we had and still<br />
have to deal with several questions concering the choice of BioBricks,<br />
genes and vectors to construct a firm method to determine "pollution" by<br />
steroids. As a conclusion, we have to meet two major requirements for<br />
our system:<br />
<br />
* It should be as cost-efficient as possible for easy and regular<br />
application<br />
* It should be resistant to yeast's own metabolism (Not be disturbed by<br />
unexpected occuring expression).<br />
<br />
At first we had to find an appropriate receptor to "grab" steroid<br />
hormones in efluents. This should fulfill the following requirements:<br />
<br />
* It should only be responsible to substrates we wish to detect, so the<br />
results of the test will not be falsified.<br />
* It has to be easiliy integrated into yeast's cell membrane.<br />
* Its nucleic acid sequence should not be too long so we can put it onto<br />
a plasmid vector.<br />
<br />
We chose the membrane progesterone receptor of the zebra fish (''Danio<br />
rerio'') and the African clawed frog (''Xenopus laevis''). We focused on<br />
these receptors, since they are easy to duplicate and interact with a<br />
broad bandwith of sex-determining hormones.<br />
<br />
The next step was to select an appropriate organism to express these<br />
receptors. After some research, we could narrow our options down to two<br />
organisms:<br />
<br />
* Escherichia coli<br />
* Saccharomyces cerevisiae<br />
<br />
Finally we decided for yeast, since it has been done more research with<br />
it according to our prefered receptors. In addition yeast is an<br />
eukaryote making it more easier to integrate mPRs into their cell<br />
membrane.<br />
<br />
== '''Mechanism''' ==<br />
<br />
[[File:Igem_2.3.jpg|thumb|right|Planned mechanism]]<br />
<br />
Naturally occuring iron receptors of the PAQR family are found to block<br />
FET3 promotor on high level of extra cellular iron. According to<br />
[http://www.ncbi.nlm.nih.gov/pubmed/18603275 Smith JL et al] expressed<br />
human mPR in yeast induced the same signal ligating to estrogen. Relying<br />
on this results we will try to express various mPRs of ''Danio rerio'',<br />
''Xanophus laevis'' in yeast which we find better fitting to measure<br />
endocrine substances that influence fish.<br />
<br />
We will transform the negative signal of FET3 by letting it control an<br />
inhibitor (ROX1 and MIG1). This inhibitor will regulate the promotor of<br />
our reporter gene. For the promoter of our reporter gene we chose ANB1<br />
and SUC2, because these are targets of ROX1 and MIG1. By negating the<br />
negative signal of FET3 we hope to obtain a positive signal sensitive<br />
enough to measure low concentrations of different endocrine substances.<br />
<br />
=== Receptors ===<br />
<br />
Membrane bound, 7-Transmembranereceptor (C-terminus inside,N-terminus<br />
outside), PAQR family (progesterone adiponectin Q receptor), Hly-III<br />
superfamily G-Protein coupled: activation of inhibitori Gi units:<br />
reduced adenylyl-cyclase activity. <br />
<br />
=== Inhibitors and their targets ===<br />
<br />
An appropriate inhibitor/promotor combination is a crucial step in our<br />
pathway and should be selected wisely.<br />
<br />
Finally we chose FET3 as our promoter and both ROX1 and MIG1 as<br />
inhibitors. It is very likely, that they don't seem to repress any gene<br />
expression that are crucial for yeast.<br />
<br />
=== Reporter genes ===<br />
<br />
The last station of our signaling pathway should be a reporter gene<br />
which amplifies our initial signal to allow a quantitative measurement.<br />
<br />
The enzyme '''luciferase''' fulfills these conditions and is our<br />
candidate of choice.<br />
<br />
== Implementation ==<br />
<br />
<br />
=== Promoter ===<br />
[[File:PRS313_Padh1_Danio_Tadh1.png|promoter1]]<br />
[[File:PRS313_Padh1_Xenopus_Tadh1.png|promoter2]]<br />
=== Inhibitor ===<br />
[[File:PRS315_Pfet3_rox1_Tadh1_V3.png|inhibitor1]]<br />
[[File:pRS315_Pfet3_mig1_Tadh1.png|inhibitor2]]<br />
=== Reporter ===<br />
[[File:PRS316_Panb1_lacZ_Tadh1.png|reporter1]]<br />
[[File:pRS316_Psuc2_lacZ_Tadh1.png|reporter2]]<br />
[[File:PRS316_Psuc2_Luciferase_Tadh1.png|reporter3]]<br />
<br />
== Measurement ==<br />
<br />
The measurement itself should be limited to an optical one. With this<br />
idea in mind, we decided for reporter genes like lacZ and luciferase,<br />
because these produce signals which can simply be quantified by optical<br />
measurement methods.<br />
<br />
== References ==<br />
<br />
* [1] Jessica L. Smith, Brian R. Kupchak, Ibon Garitaonandia, L. Kim Hoang, Andrew S. Maina, Lisa M. Regalla, and Thomas J. Lyons - 2008 - Heterologous expression of human mPRα, mPRβ and mPRγ in yeast confirms their ability to function as membrane progesterone receptors<br />
* [2] Sumpter, Johnson (2008) Reflections on endocrine disruption in the aquatic environment<br />
<!--<br />
* Liew et. al. - 2012 Polygenic Sex Determination System in Zebrafish<br />
* Hanna et al. - 2006 - Cell-surface expression, progestin binding, and<br />
rapid nongenomic signaling of zebrafish membrane progestin receptors<br />
alpha and beta in transfected cells<br />
* Reupke - 2011 - Detektion dopingrelevanter anaboler Steroide in<br />
Pferdeurin und Pferdeplasma mithilfe eines Reportergen-Assays in<br />
Hefezellen. (Detection of doping relevant anabolic steroids in horse<br />
urin and blood plasma using yeast reporter gene assays)<br />
* “Assault on the male”, BBC 1996. Video report<br />
http://www.youtube.com/watch?v=LkxIJJI37bQ<br />
* Dr. Volker Scheil, The impact of potential environmental stressors on<br />
early development and cellular and biochemical biomarkers in fish,<br />
Main research: Fish embryotoxicity, histopathology and stress protein<br />
(hsp 70) responses.<br />
* Harris et al. - 2011 - The consequences of feminization in breeding<br />
groups of wild fish<br />
* Liew et al. - 2012 - Polygenic Sex Determination System in Zebrafish<br />
* Sumpter, Johnson - 2008 - 10th Anniversary Perspective Reflections on<br />
endocrine disruption in the aquatic environment from known knowns to<br />
unknown unknowns (and many things in between)<br />
* Werner, Palace, Wautier - 2006 - Reproductive fitness of lake trout<br />
(Salvelinus namaycush) exposed to environmentally relevant<br />
concentrations of the potent estrogen ethynylestradiol (EE2) in a<br />
whole lake exposure experiment<br />
* Fenske, M., Maack, G., Schäfers, C., & Segner, H. (2005). An<br />
environmentally relevant concentration of estrogen induces arrest of<br />
male gonad development in zebrafish, Danio rerio. Environmental<br />
toxicology and chemistry / SETAC, 24(5), 1088-98. Retrieved from<br />
http://www.ncbi.nlm.nih.gov/pubmed/16110986 (kein PDF)<br />
* Schäfers, C., Teigeler, M., Wenzel, A., Maack, G., Fenske, M., &<br />
Segner, H. (2007). Concentration- and time-dependent effects of the<br />
synthetic estrogen, 17alpha-ethinylestradiol, on reproductive<br />
capabilities of the zebrafish, Danio rerio. Journal of toxicology and<br />
environmental health. Part A, 70(9), 768-79.<br />
doi:10.1080/15287390701236470<br />
* Segner, H., Caroll, K., Fenske, M., Janssen, C. R., Maack, G., Pascoe,<br />
D., Schäfers, C., et al. (2003). Identification of<br />
endocrine-disrupting effects in aquatic vertebrates and invertebrates:<br />
report from the European IDEA project. Ecotoxicology and environmental<br />
safety, 54(3), 302-14. Retrieved from<br />
http://www.ncbi.nlm.nih.gov/pubmed/12651186 (kein PDF)<br />
* Hanna et al. - 2006 - Cell-surface expression, progestin binding, and<br />
rapid nongenomic signaling of zebrafish membrane progestin receptors<br />
alpha and beta in transfected cells<br />
* Thomas et al. - 2007 - Steroid and G protein binding characteristics<br />
of the seatrout and human progestin membrane receptor alpha subtypes<br />
and their evolutionary origins<br />
* Thomas - 2008 - Characteristics of membrane progestin receptor alpha<br />
(mPR ) and progesterone membrane receptor component 1 (PGMRC1) and<br />
their roles in mediating rapid progestin actions<br />
* Jessica L. Smith, Brian R. Kupchak, Ibon Garitaonandia, L. Kim Hoang,<br />
Andrew S. Maina, Lisa M. Regalla, and Thomas J. *Lyons - 2008 -<br />
Heterologous expression of human mPRα, mPRβ and mPRγ in yeast confirms<br />
their ability to function as membrane progesterone receptors<br />
<br />
--></div>RoMannhttp://2012.igem.org/Team:Tuebingen/SafetyTeam:Tuebingen/Safety2012-09-04T12:57:12Z<p>RoMann: /* Safety */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
<br />
== Safety ==<br />
<br />
''Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?''<br />
<br />
All our work is supervised by PhD students, PhDs and professors of our university who are very experienced in lab work and are currently working in genetics. We document our work in our lab notebook to enable replicability and prevent unnecessary errors.<br />
<br />
Our project does not pose any dangers to researchers or the public: We are following widely used lab protocols in a appropriate lab facility. The organisms used in our project, namely ''E. coli'' (TOP10 strain) and ''S. cerevisiae'', are well-known and considered safe.<br />
<br />
Although, by building a biosensor we are tackling an environmental problem, our system is not intended to be used in the field but in a lab only.<br />
<br />
<br />
''Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?''<br />
<br />
We have designed 3 different types of BioBricks. Membrane bound receptors for sensing hormones, a small inverter system to regulate our reporter gene. None of these are found toxic or dangerous by any means.<br />
The receptors we use are synthesized after genome-sequences of ''Danio rerio'' and ''Xenopus laevis'', both are commonly used model organisms. The rest of the genes and promoters are extracted from the ''Saccharomyces cerevisiae'' genome via PCR. All of the parts we use are expressed in wildtype organisms and not known to have any harmful effect on humans or other organisms.<br />
<br />
<br />
''Which specific biosafety rules or guidelines do you have to consider in your country?''<br />
<br />
In Germany any work with genetically modified organisms is regulated by the "Gentechnikgesetz". There are different biosafety levels reaching from 1 to 4. Our lab has been registered as level 1.<br />
Our work matches the definition of level 1 since on our current understanding we see no threats on human health or the environment.<br />
<br />
Genetically modified organisms are allowed to be released into nature only on permission of the Umweltministerium and once freed need to be constantly monitored.<br />
<br />
<br />
''Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?''<br />
<br />
BioBricks are basically DNA-Sequences that are not dangerous to the environment until they are expressed in a proper organism. <br />
At first you have to take care that no cell culture with this BioBrick in its genome leaves the lab. But we have to consider this case as well. That means we have to take care that standard organisms are not able to express the parts outside of the lab. <br />
It might be possible to add some kind of self-destructive header sequence to the part which ensures, that the concerned cells get destroyed when they try to translate the parts into proteins. Some kind of induced cell apoptosis or nekrosis might be used.</div>RoMannhttp://2012.igem.org/Team:Tuebingen/ProjectTeam:Tuebingen/Project2012-05-25T17:45:37Z<p>RoMann: /* Overview */</p>
<hr />
<div>{{Template:Tuebingen2012navigation}}<br />
__NOTOC__<br />
== '''Overview''' ==<br />
Our general aim is to establish a simple mechanism which allows us to measure an estrogen concentration in a solution as precisely as possible. Therefore we want to clone the membrane progestin receptor (mPR) of the zebrafish (''Danio rerio'') in yeast (''saccharomyces cerevisiae'') and link it to a reporter gene, which allows a quantitative measurement.<br />
<br />
== '''Motivation''' ==<br />
Why do we want to establish a mechanism for steroid measurement?<br />
<br />
Steroid hormones, especially estrogens, occur in all vertebrates and play a crucial role in sexual differentiation. In recent times the pollution of waters with these hormones has become an increasing problem for the aquatic fauna. <br />
Particularly waters functionalized by humans or adjacent to human settlements, for example in areas with agricultural use, show increased estrogen-concentrations.<br />
<br />
Scientific studies based on ''Danio rerio'' showed that the consequences are devastating. High concentrations of 17α-ethinylestradiol, a hormone in most birth-control pills, affected the sex differentiation of ''Danio rerio'' leading to development of ovotestis or complete feminization (Andersenc, 2002).<br />
Intersex-fish have been reported in UK rivers since 1978 just downstream of an sewage treatment plant.<br />
<br />
We believe that a first step in finding a solution to this environmental problem is an accurate and reliable method to quantify steroids which is easy to use and limited to an optical measurement.<br />
<br />
== '''Mechanism''' ==<br />
<br />
=== '''Receptors''' ===<br />
<br />
Membrangebunden, 7-Transmembranrezeptor (Innen C-Terminus, außen N-Terminus), PAQR-Familie (progesterone adiponectin Q receptor), Hly-III superfamily<br />
G-Protein gekoppelt: Aktivierung von inhibitorische Gi-Einheiten: Verringerung der Aktivität der Adenylyl-Cyclase <br />
G Proteins (Protein Data Bank) Artikel zu G-Proteinen - Struktur und Funktionsweise, schöne Bilder<br />
<br />
Sequenz mPR alpha (Zebrafisch): [http://www.ncbi.nlm.nih.gov/nuccore/AY149121.1 NCBI],<br />
<br />
Sequenz mPR beta (Zebrafisch): [http://www.ncbi.nlm.nih.gov/nuccore/AY149120.1 NCBI]<br />
<br />
Homologe zum mPR Alpha Danio rerio:<br />
*Goldfisch, Carassius auratus<br />
*Katzenwels, Ictalurus punctatus<br />
*Hundszungen, Cynoglossus semilaevis<br />
*Flunder, Paralichthys lethostigma<br />
*Meerforelle, Cynoscion nebulosus<br />
*Buntbarsch, Oreochromis niloticus<br />
*Umberfisch, Micropogonias undulatus<br />
<br />
Zebrafrisch (Danio rerio)<br />
In Tübingen:<br />
*Tübingen Map of the [http://wwwmap.tuebingen.mpg.de/ Zebrafish Genome]<br />
*[http://www.mnf.uni-tuebingen.de/fachbereiche/biologie/institute/evolutionecology/lehrbereiche/physiologische-oekologie-der-tiere/staff/volker-scheil.html Dr. Volker Scheil], The impact of potential environmental stressors on early development and cellular and biochemical biomarkers in fish, Main research: Fish embryotoxicity, histopathology and stress protein (hsp 70) responses.<br />
<br />
=== '''Inhibitor and promotor''' ===<br />
<br />
=== '''Reporter gene''' ===<br />
<br />
== '''Measurement''' ==<br />
<br />
== '''References''' ==<br />
<br />
=== Sumpter, Johnson (2008) Reflections on endocrine disruption in the aquatic environment ===<br />
*Diskussion: Wurde schon zuviel Geld, Mühe und Arbeit in die Erforschung von endokriner Disruption gesteckt?<br />
*Die meisten Mensche erachten “hormonelle Verschmutzung” als geringfügiges Problem.<br />
*Erste Beobachtung von intersexuellen Fischen (roach - Rotauge - Rutilis rutilis) 1978 in England stromabwärts einer Kläranlage.<br />
*Vorkommen von intersexuellen Fischen abhängig vom Alter.<br />
*Erste Studien zeigen einen kummulativen Effekt der endokrinen Substanzen.<br />
*[http://de.wikipedia.org/wiki/Vitellogenine#Verwendung_als_Estrogen-Test Vitellogenin] als indikator für feminisierung/oestrogene. Nach zwei Wochen Abwasser -> 100.000-fache Konzentration in männlichen Fischen.<br />
*Mitte der 90er: Intersexuelle Fische in ganz England verbreitet.<br />
*Studien belegen den Verdacht auf den Einfluss durch [http://de.wikipedia.org/wiki/Estron Estron], [http://de.wikipedia.org/wiki/Estradiol Estradiol] (natürlich) und [http://de.wikipedia.org/wiki/Estradiol Ethinylestradiol] (synthetisch). Effekt im niedrigen ng/Liter bereich<br />
*“male suppressing/female enhancing effect”<br />
*Ebenfalls gemessene [http://de.wikipedia.org/wiki/Alkylophenol Alkylphenole], insbesondere [http://de.wikipedia.org/wiki/Nonylphenol Nonylphenol] und Oktylphenol haben östrogene Wirkung. Die benutzung dieser Stoffklasse ist in Europa bereits eingeschränkt, in den [http://water.epa.gov/scitech/swguidance/standards/criteria/aqlife/pollutants/nonylphenol/nonylphenol-fs.cfm USA] noch nicht.<br />
*Östrogene beeinflussen die Expression Tausender von Genen<br />
*Der Substanzmix im Gewässer erschwert die Detektion und Analyse: “it has proved extremely difficult to accurately determine the concentration of steroid oestrogens in effluents”<br />
<br />
=== Liew et. al. - 2012 Polygenic Sex Determination System in Zebrafish (Polygenes Geschlechtsbestimmungssystem) ===<br />
*Zebrafish sex is genetically determined with limited, secondary influences from the environment.<br />
*No sign for CSD (chromosomal sex determination) was found in the species, which results in the conclusion, that Zebrafish might has a polygenic sex determination system. <br />
*experiments were performed at Max-Planck-Institut für Entwicklungsbiologie in Tübingen, the zebrafish of different strains were bought from a local aquarium shop<br />
*genomic DNA samples were extracted from tail fins<br />
*“only” 89% of the DNA was restored<br />
*there are no substantial differences between zebrafish male and female genomes and no dominant male/female genomes were discovered<br />
*lack of oxygen level causes a reduction of estrogen synthesis → more androgen was built, which led to more male zebrafish in the population<br />
<br />
=== Hanna et al. - 2006 - Cell-surface expression, progestin binding, and rapid nongenomic signaling of zebrafish membrane progestin receptors alpha and beta in transfected cells ===<br />
*Ergebnis der Studie: spezifische Aktivierung der Progestin Receptoren mPRa und mPRb durch Steroide (Progesteron, Cortisol, Testosteron, Estrogen, 11-Desoxycortisol) im Zebrafisch <br />
-> MAPK (mitogen-activated ptotein kinase) Aktivierung bei gleichzeitigem Absenken der Adenylatcyclase Aktivität (über Pertussis Toxin-sensitiven Gi Pathway)<br />
*Expression der der Rezeptoren in Säugetierbrustkrebszellen<br />
*Überprüfung der Expression und Lokalisation der Rezeptoren mit <br />
**Western blot<br />
**Flow cytometry<br />
**biotin surface labeling<br />
<br />
=== Reupke - 2011 - Detektion dopingrelevanter anaboler Steroide in Pferdeurin und Pferdeplasma mithilfe eines Reportergen-Assays in Hefezellen ===<br />
*Hefe mit dem humanen Androgenrezeptor hAR (fest ins Genom integriert) und dem Reportergen LacZ (-> ß-Galactosidase)<br />
*Rezeptor liegt allerdings nur im Cytoplasma vor, ist nicht membranstänig -> Problem, da Steroide in konjugierter Form nicht durch die ZM diffundieren können, aber im Urin sehr häuftig in dieser Form vorliegen<br />
*-> weitere potentielle Anwendung für unser Projekt: Dopingtest für Anabolika? Wenn unsere Hefe mit unserem Rezeptor so funktioniert, wie wir uns es vorstellen, wäre das eine deutliche Verbesserung der in der Doktorarbeit beschriebenen Methode, Einsatz auch im humanen Leistungssport denkbar.<br />
*-> evt. interessant für Sponsoren (Deutscher Olympischer Sport Bund, Nationale Anti Doping Agentur, ect.)<br />
<br />
=== Jessica L. Smith, Brian R. Kupchak, Ibon Garitaonandia, L. Kim Hoang, Andrew S. Maina, Lisa M. Regalla, and Thomas J. Lyons - 2008 - Heterologous expression of human mPRα, mPRβ and mPRγ in yeast confirms their ability to function as membrane progesterone receptors ===<br />
*menschliche mPRa, PRb, mPRg der Familie der Progestin und AdipoQ Rezeptoren (PAQR) wurden in Hefe exprimiert <br />
*man wollte die genaue Rolle dieser Rezeptoren innerhalb des nicht genomischen Progesteron Signalweges herausfinden<br />
*Bei Bindung von Liganden an die PAQR-Rezeptoren in Hefe wurde die FET3 Expression aktiviert <br />
**-> mPRa, mPRb, mPRg hemmen allerdings die FET3 Expression<br />
**-> die Expression von FET3 wird normalerweise durch den Osmotin Rezeptor in Hefe kontrolliert<br />
**-> FET3 wurde somit als Reporter für die Funktionalität der PAQR-Rezeptoren verwendet<br />
<br />
<br />
*“Assault on the male”, BBC 1996. Dokumentation zum Thema http://www.youtube.com/watch?v=LkxIJJI37bQ<br />
*Dr. Volker Scheil, The impact of potential environmental stressors on early development and cellular and biochemical biomarkers in fish, Main research: Fish embryotoxicity, histopathology and stress protein (hsp 70) responses.<br />
*Harris et al. - 2011 - The consequences of feminization in breeding groups of wild fish<br />
*Liew et al. - 2012 - Polygenic Sex Determination System in Zebrafish<br />
*Sumpter, Johnson - 2008 - 10th Anniversary Perspective Reflections on endocrine disruption in the aquatic environment from known knowns to unknown unknowns (and many things in between)<br />
*Werner, Palace, Wautier - 2006 - Reproductive fitness of lake trout (Salvelinus namaycush) exposed to environmentally relevant concentrations of the potent estrogen ethynylestradiol (EE2) in a whole lake exposure experiment<br />
*Fenske, M., Maack, G., Schäfers, C., & Segner, H. (2005). An environmentally relevant concentration of estrogen induces arrest of male gonad development in zebrafish, Danio rerio. Environmental toxicology and chemistry / SETAC, 24(5), 1088-98. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/16110986 (kein PDF)<br />
*Schäfers, C., Teigeler, M., Wenzel, A., Maack, G., Fenske, M., & Segner, H. (2007). Concentration- and time-dependent effects of the synthetic estrogen, 17alpha-ethinylestradiol, on reproductive capabilities of the zebrafish, Danio rerio. Journal of toxicology and environmental health. Part A, 70(9), 768-79. doi:10.1080/15287390701236470<br />
*Segner, H., Caroll, K., Fenske, M., Janssen, C. R., Maack, G., Pascoe, D., Schäfers, C., et al. (2003). Identification of endocrine-disrupting effects in aquatic vertebrates and invertebrates: report from the European IDEA project. Ecotoxicology and environmental safety, 54(3), 302-14. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/12651186 (kein PDF)<br />
*Hanna et al. - 2006 - Cell-surface expression, progestin binding, and rapid nongenomic signaling of zebrafish membrane progestin receptors alpha and beta in transfected cells<br />
*Thomas et al. - 2007 - Steroid and G protein binding characteristics of the seatrout and human progestin membrane receptor alpha subtypes and their evolutionary origins<br />
*Thomas - 2008 - Characteristics of membrane progestin receptor alpha (mPR ) and progesterone membrane receptor component 1 (PGMRC1) and their roles in mediating rapid progestin actions<br />
*Jessica L. Smith, Brian R. Kupchak, Ibon Garitaonandia, L. Kim Hoang, Andrew S. Maina, Lisa M. Regalla, and Thomas J. *Lyons - 2008 - Heterologous expression of human mPRα, mPRβ and mPRγ in yeast confirms their ability to function as membrane progesterone receptors</div>RoMannhttp://2012.igem.org/Team:Tuebingen/ProjectTeam:Tuebingen/Project2012-05-25T17:41:41Z<p>RoMann: /* Motivation */</p>
<hr />
<div>{{Template:Tuebingen2012navigation}}<br />
__NOTOC__<br />
== '''Overview''' ==<br />
Our general aim is to establish a mechanism which allows us to define precisely a estrogen concentration in a dilute solution. Therefore we want to introduce the membrane progestin receptor (mPR) of the zebrafish (Danio rerio) in yeast (saccharomyces cerevisiae), linked to a reporter gene, which should allow us a quantitative measurement.<br />
<br />
== '''Motivation''' ==<br />
Why do we want to establish a mechanism for steroid measurement?<br />
<br />
Steroid hormones, especially estrogens, occur in all vertebrates and play a crucial role in sexual differentiation. In recent times the pollution of waters with these hormones has become an increasing problem for the aquatic fauna. <br />
Particularly waters functionalized by humans or adjacent to human settlements, for example in areas with agricultural use, show increased estrogen-concentrations.<br />
<br />
Scientific studies based on ''Danio rerio'' showed that the consequences are devastating. High concentrations of 17α-ethinylestradiol, a hormone in most birth-control pills, affected the sex differentiation of ''Danio rerio'' leading to development of ovotestis or complete feminization (Andersenc, 2002).<br />
Intersex-fish have been reported in UK rivers since 1978 just downstream of an sewage treatment plant.<br />
<br />
We believe that a first step in finding a solution to this environmental problem is an accurate and reliable method to quantify steroids which is easy to use and limited to an optical measurement.<br />
<br />
== '''Mechanism''' ==<br />
<br />
=== '''Receptors''' ===<br />
<br />
Membrangebunden, 7-Transmembranrezeptor (Innen C-Terminus, außen N-Terminus), PAQR-Familie (progesterone adiponectin Q receptor), Hly-III superfamily<br />
G-Protein gekoppelt: Aktivierung von inhibitorische Gi-Einheiten: Verringerung der Aktivität der Adenylyl-Cyclase <br />
G Proteins (Protein Data Bank) Artikel zu G-Proteinen - Struktur und Funktionsweise, schöne Bilder<br />
<br />
Sequenz mPR alpha (Zebrafisch): [http://www.ncbi.nlm.nih.gov/nuccore/AY149121.1 NCBI],<br />
<br />
Sequenz mPR beta (Zebrafisch): [http://www.ncbi.nlm.nih.gov/nuccore/AY149120.1 NCBI]<br />
<br />
Homologe zum mPR Alpha Danio rerio:<br />
*Goldfisch, Carassius auratus<br />
*Katzenwels, Ictalurus punctatus<br />
*Hundszungen, Cynoglossus semilaevis<br />
*Flunder, Paralichthys lethostigma<br />
*Meerforelle, Cynoscion nebulosus<br />
*Buntbarsch, Oreochromis niloticus<br />
*Umberfisch, Micropogonias undulatus<br />
<br />
Zebrafrisch (Danio rerio)<br />
In Tübingen:<br />
*Tübingen Map of the [http://wwwmap.tuebingen.mpg.de/ Zebrafish Genome]<br />
*[http://www.mnf.uni-tuebingen.de/fachbereiche/biologie/institute/evolutionecology/lehrbereiche/physiologische-oekologie-der-tiere/staff/volker-scheil.html Dr. Volker Scheil], The impact of potential environmental stressors on early development and cellular and biochemical biomarkers in fish, Main research: Fish embryotoxicity, histopathology and stress protein (hsp 70) responses.<br />
<br />
=== '''Inhibitor and promotor''' ===<br />
<br />
=== '''Reporter gene''' ===<br />
<br />
== '''Measurement''' ==<br />
<br />
== '''References''' ==<br />
<br />
=== Sumpter, Johnson (2008) Reflections on endocrine disruption in the aquatic environment ===<br />
*Diskussion: Wurde schon zuviel Geld, Mühe und Arbeit in die Erforschung von endokriner Disruption gesteckt?<br />
*Die meisten Mensche erachten “hormonelle Verschmutzung” als geringfügiges Problem.<br />
*Erste Beobachtung von intersexuellen Fischen (roach - Rotauge - Rutilis rutilis) 1978 in England stromabwärts einer Kläranlage.<br />
*Vorkommen von intersexuellen Fischen abhängig vom Alter.<br />
*Erste Studien zeigen einen kummulativen Effekt der endokrinen Substanzen.<br />
*[http://de.wikipedia.org/wiki/Vitellogenine#Verwendung_als_Estrogen-Test Vitellogenin] als indikator für feminisierung/oestrogene. Nach zwei Wochen Abwasser -> 100.000-fache Konzentration in männlichen Fischen.<br />
*Mitte der 90er: Intersexuelle Fische in ganz England verbreitet.<br />
*Studien belegen den Verdacht auf den Einfluss durch [http://de.wikipedia.org/wiki/Estron Estron], [http://de.wikipedia.org/wiki/Estradiol Estradiol] (natürlich) und [http://de.wikipedia.org/wiki/Estradiol Ethinylestradiol] (synthetisch). Effekt im niedrigen ng/Liter bereich<br />
*“male suppressing/female enhancing effect”<br />
*Ebenfalls gemessene [http://de.wikipedia.org/wiki/Alkylophenol Alkylphenole], insbesondere [http://de.wikipedia.org/wiki/Nonylphenol Nonylphenol] und Oktylphenol haben östrogene Wirkung. Die benutzung dieser Stoffklasse ist in Europa bereits eingeschränkt, in den [http://water.epa.gov/scitech/swguidance/standards/criteria/aqlife/pollutants/nonylphenol/nonylphenol-fs.cfm USA] noch nicht.<br />
*Östrogene beeinflussen die Expression Tausender von Genen<br />
*Der Substanzmix im Gewässer erschwert die Detektion und Analyse: “it has proved extremely difficult to accurately determine the concentration of steroid oestrogens in effluents”<br />
<br />
=== Liew et. al. - 2012 Polygenic Sex Determination System in Zebrafish (Polygenes Geschlechtsbestimmungssystem) ===<br />
*Zebrafish sex is genetically determined with limited, secondary influences from the environment.<br />
*No sign for CSD (chromosomal sex determination) was found in the species, which results in the conclusion, that Zebrafish might has a polygenic sex determination system. <br />
*experiments were performed at Max-Planck-Institut für Entwicklungsbiologie in Tübingen, the zebrafish of different strains were bought from a local aquarium shop<br />
*genomic DNA samples were extracted from tail fins<br />
*“only” 89% of the DNA was restored<br />
*there are no substantial differences between zebrafish male and female genomes and no dominant male/female genomes were discovered<br />
*lack of oxygen level causes a reduction of estrogen synthesis → more androgen was built, which led to more male zebrafish in the population<br />
<br />
=== Hanna et al. - 2006 - Cell-surface expression, progestin binding, and rapid nongenomic signaling of zebrafish membrane progestin receptors alpha and beta in transfected cells ===<br />
*Ergebnis der Studie: spezifische Aktivierung der Progestin Receptoren mPRa und mPRb durch Steroide (Progesteron, Cortisol, Testosteron, Estrogen, 11-Desoxycortisol) im Zebrafisch <br />
-> MAPK (mitogen-activated ptotein kinase) Aktivierung bei gleichzeitigem Absenken der Adenylatcyclase Aktivität (über Pertussis Toxin-sensitiven Gi Pathway)<br />
*Expression der der Rezeptoren in Säugetierbrustkrebszellen<br />
*Überprüfung der Expression und Lokalisation der Rezeptoren mit <br />
**Western blot<br />
**Flow cytometry<br />
**biotin surface labeling<br />
<br />
=== Reupke - 2011 - Detektion dopingrelevanter anaboler Steroide in Pferdeurin und Pferdeplasma mithilfe eines Reportergen-Assays in Hefezellen ===<br />
*Hefe mit dem humanen Androgenrezeptor hAR (fest ins Genom integriert) und dem Reportergen LacZ (-> ß-Galactosidase)<br />
*Rezeptor liegt allerdings nur im Cytoplasma vor, ist nicht membranstänig -> Problem, da Steroide in konjugierter Form nicht durch die ZM diffundieren können, aber im Urin sehr häuftig in dieser Form vorliegen<br />
*-> weitere potentielle Anwendung für unser Projekt: Dopingtest für Anabolika? Wenn unsere Hefe mit unserem Rezeptor so funktioniert, wie wir uns es vorstellen, wäre das eine deutliche Verbesserung der in der Doktorarbeit beschriebenen Methode, Einsatz auch im humanen Leistungssport denkbar.<br />
*-> evt. interessant für Sponsoren (Deutscher Olympischer Sport Bund, Nationale Anti Doping Agentur, ect.)<br />
<br />
=== Jessica L. Smith, Brian R. Kupchak, Ibon Garitaonandia, L. Kim Hoang, Andrew S. Maina, Lisa M. Regalla, and Thomas J. Lyons - 2008 - Heterologous expression of human mPRα, mPRβ and mPRγ in yeast confirms their ability to function as membrane progesterone receptors ===<br />
*menschliche mPRa, PRb, mPRg der Familie der Progestin und AdipoQ Rezeptoren (PAQR) wurden in Hefe exprimiert <br />
*man wollte die genaue Rolle dieser Rezeptoren innerhalb des nicht genomischen Progesteron Signalweges herausfinden<br />
*Bei Bindung von Liganden an die PAQR-Rezeptoren in Hefe wurde die FET3 Expression aktiviert <br />
**-> mPRa, mPRb, mPRg hemmen allerdings die FET3 Expression<br />
**-> die Expression von FET3 wird normalerweise durch den Osmotin Rezeptor in Hefe kontrolliert<br />
**-> FET3 wurde somit als Reporter für die Funktionalität der PAQR-Rezeptoren verwendet<br />
<br />
<br />
*“Assault on the male”, BBC 1996. Dokumentation zum Thema http://www.youtube.com/watch?v=LkxIJJI37bQ<br />
*Dr. Volker Scheil, The impact of potential environmental stressors on early development and cellular and biochemical biomarkers in fish, Main research: Fish embryotoxicity, histopathology and stress protein (hsp 70) responses.<br />
*Harris et al. - 2011 - The consequences of feminization in breeding groups of wild fish<br />
*Liew et al. - 2012 - Polygenic Sex Determination System in Zebrafish<br />
*Sumpter, Johnson - 2008 - 10th Anniversary Perspective Reflections on endocrine disruption in the aquatic environment from known knowns to unknown unknowns (and many things in between)<br />
*Werner, Palace, Wautier - 2006 - Reproductive fitness of lake trout (Salvelinus namaycush) exposed to environmentally relevant concentrations of the potent estrogen ethynylestradiol (EE2) in a whole lake exposure experiment<br />
*Fenske, M., Maack, G., Schäfers, C., & Segner, H. (2005). An environmentally relevant concentration of estrogen induces arrest of male gonad development in zebrafish, Danio rerio. Environmental toxicology and chemistry / SETAC, 24(5), 1088-98. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/16110986 (kein PDF)<br />
*Schäfers, C., Teigeler, M., Wenzel, A., Maack, G., Fenske, M., & Segner, H. (2007). Concentration- and time-dependent effects of the synthetic estrogen, 17alpha-ethinylestradiol, on reproductive capabilities of the zebrafish, Danio rerio. Journal of toxicology and environmental health. Part A, 70(9), 768-79. doi:10.1080/15287390701236470<br />
*Segner, H., Caroll, K., Fenske, M., Janssen, C. R., Maack, G., Pascoe, D., Schäfers, C., et al. (2003). Identification of endocrine-disrupting effects in aquatic vertebrates and invertebrates: report from the European IDEA project. Ecotoxicology and environmental safety, 54(3), 302-14. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/12651186 (kein PDF)<br />
*Hanna et al. - 2006 - Cell-surface expression, progestin binding, and rapid nongenomic signaling of zebrafish membrane progestin receptors alpha and beta in transfected cells<br />
*Thomas et al. - 2007 - Steroid and G protein binding characteristics of the seatrout and human progestin membrane receptor alpha subtypes and their evolutionary origins<br />
*Thomas - 2008 - Characteristics of membrane progestin receptor alpha (mPR ) and progesterone membrane receptor component 1 (PGMRC1) and their roles in mediating rapid progestin actions<br />
*Jessica L. Smith, Brian R. Kupchak, Ibon Garitaonandia, L. Kim Hoang, Andrew S. Maina, Lisa M. Regalla, and Thomas J. *Lyons - 2008 - Heterologous expression of human mPRα, mPRβ and mPRγ in yeast confirms their ability to function as membrane progesterone receptors</div>RoMann