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Team:UTK-Knoxville/Aer
From 2013.igem.org
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</div> | </div> | ||
<h2><span class="mw-headline">Aer</span></h2> | <h2><span class="mw-headline">Aer</span></h2> | ||
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<p><img src="https://static.igem.org/mediawiki/2013/thumb/4/48/Aer_plasmid.png/642px-Aer_plasmid.png" alt="Aer plasmid" width="642" height="599" border="0" usemap="#pBR322" id="sprytrigger1"> | <p><img src="https://static.igem.org/mediawiki/2013/thumb/4/48/Aer_plasmid.png/642px-Aer_plasmid.png" alt="Aer plasmid" width="642" height="599" border="0" usemap="#pBR322" id="sprytrigger1"> | ||
<map name="pBR322"> | <map name="pBR322"> | ||
<area shape="poly" coords="87,301" href="#"> | <area shape="poly" coords="87,301" href="#"> | ||
| - | <area shape="poly" coords="66, | + | <area shape="poly" coords="66,275,79,215,103,163,131,121,164,90,197,71,222,57,255,44,261,62,246,66,208,82,177,107,147,137,119,175,97,225,84,275" href="https://2013.igem.org/Team:UTK-Knoxville/Aer#KanRDETAILS" alt="KanR"> |
| - | <area shape="poly" coords="344,36,341,56,374,60,378,41" href="https://2013.igem.org/Team:UTK-Knoxville/ | + | <area shape="poly" coords="344,36,341,56,374,60,378,41" href="https://2013.igem.org/Team:UTK-Knoxville/Aer#T7promoterDETAILS" alt="T7"> |
| - | <area shape="poly" coords="542,203,560,198,548,173,528,140,502,114,478,90,457,73,409,54,375,43,373,61,427,81,472,106,501,136,529,175" href="https://2013.igem.org/Team:UTK-Knoxville/ | + | <area shape="poly" coords="542,203,560,198,548,173,528,140,502,114,478,90,457,73,409,54,375,43,373,61,427,81,472,106,501,136,529,175" href="https://2013.igem.org/Team:UTK-Knoxville/Aer#OmpRDETAILS" alt="OmpR"> |
| - | <area shape="poly" coords="549,213,562,210,571,239,574,272,577,303,573,334,562,375,552,401,539,426,519,455,504,471,489,458,513,432,531,404,550,367,557,327,560,285,559,261" href="https://2013.igem.org/Team:UTK-Knoxville/ | + | <area shape="poly" coords="549,213,562,210,571,239,574,272,577,303,573,334,562,375,552,401,539,426,519,455,504,471,489,458,513,432,531,404,550,367,557,327,560,285,559,261" href="https://2013.igem.org/Team:UTK-Knoxville/Aer#AerDETAILS" alt="Aer"> |
| - | <area shape="poly" coords="285,524,326,527,374,522,399,514,428,502,452,488,468,478,476,470,479,467,493,479,484,487,471,498,450,511,424,526,381,540,355,544,315,545,283,544" href="https://2013.igem.org/Team:UTK-Knoxville/ | + | <area shape="poly" coords="285,524,326,527,374,522,399,514,428,502,452,488,468,478,476,470,479,467,493,479,484,487,471,498,450,511,424,526,381,540,355,544,315,545,283,544" href="https://2013.igem.org/Team:UTK-Knoxville/Aer#EnvZDETAILS" alt="EnvZ"> |
| - | <area shape="poly" coords="204,515,189,504,159,482,134,459,112,433,96,408,86,384,77,358,73,341,92,335,101,372,108,390,132,432,155,458,178,476,195,491,211,498" href="https://2013.igem.org/Team:UTK-Knoxville/ | + | <area shape="poly" coords="204,515,189,504,159,482,134,459,112,433,96,408,86,384,77,358,73,341,92,335,101,372,108,390,132,432,155,458,178,476,195,491,211,498" href="https://2013.igem.org/Team:UTK-Knoxville/Aer#pBR322DETAILS" alt="pBR322"> |
</map> | </map> | ||
</p> | </p> | ||
| + | <p> </p> | ||
| + | <h3> <span class="mw-headline" id="AerDETAILS">Aer</span></h3> | ||
| + | <p>Aer is a sensory protein that measures oxygen levels within the cell, rather than externally. When low internal oxygen levels are sensed, it will trigger reactions such as movement towards quickly oxidizable substances such as glucose or ribose. It is located in the inner membrane of the cell. Aer also regulates the expression of many genes involved in anaerobic respiration. Aer is part of the PAS domain of sensing proteins.<a name="_GoBack"></a></p> | ||
| + | <p> </p> | ||
| + | <h3> <span class="mw-headline" id="EnvZDETAILS">EnvZ</span></h3> | ||
| + | <p><u>Overview</u><br> | ||
| + | EnvZ is the histidine kinase in the EnvZ-OmpR signal transduction system, located in the inner membrane. It contains two separate transmembrane regions. EnvZ autoposphorolates, then phosphorotales the transcription factor OmpR. EnvZ and OmpR are often seen as a single, polysistronic mRNA, although there is normally much less OmpR present in any given cell.</p> | ||
| + | <p>Aer is a sensory protein that measures oxygen levels within the cell, rather than externally. When low internal oxygen levels are sensed, it will trigger reactions such as movement towards quickly oxidizable substances such as glucose or ribose. It is located in the inner membrane of the cell. Aer also regulates the expression of many genes involved in anaerobic respiration. Aer is part of the PAS domain of sensing proteins.<br> | ||
| + | We chose Aer because it is a more recently studied gene, so little is known about it. According to <a href="http://jb.asm.org/content/192/6/1740.full.pdf">Nishiyama</a> , Aer also has temperature sensing potential, so this could be another interesting aspect our project. <br> | ||
| + | Our construct consists of an Aer-EnvZ chimera protein created view overlapping extension PCR (OEPCR), and a transcription factor, OmpR. EnvZ is a histidine kinase that communicates with OmpR.</p> | ||
| + | <br clear="all"> | ||
| + | <div> | ||
| + | <p>Our platform for this construct: <br> | ||
| + | <img src="Aer_image002.jpg" alt="" width="177" height="155" border="0"><br> | ||
| + | Our construct: <br> | ||
| + | <img src="Aer_image004.jpg" alt="" width="159" height="140" border="0"></p> | ||
| + | </div> | ||
| + | <u><br clear="all"> | ||
| + | </u> | ||
| + | <div> | ||
| + | <p><u>Primers</u></p> | ||
| + | </div> | ||
| + | <br clear="all"> | ||
| + | <div> | ||
| + | <p>EnvZ_R: aaaaaactcgagttaggatccTTACCCTTCTTTTGTCGTGCC*<br> | ||
| + | OmpR_R: AAAAAACTCGAGAGGGGATCtcatgctttagagccgtccgg*<br> | ||
| + | OmpR_F: aaaaaaCATATGCAAGAGACCTACAAGATTCTG<br> | ||
| + | Aer_F: aaaaaagaattctaaagatctaaggagatataATGTCTTCTCATCCGTATGTC*<br> | ||
| + | Aer-EnvZ_R: GCGCAAGTCGTGACTTACCCatttctgacactggacacctgg*</p> | ||
| + | </div> | ||
| + | <br clear="all"> | ||
| + | <div> | ||
| + | <p>Aer-EnvZ_F: ccaggtgtccagtgtcagaaatGGGTAAGTCACGACTTGCGC*<br> | ||
| + | *Lowercase text contains a restriction site</p> | ||
| + | <p><u> </u></p> | ||
| + | <p><u>Construct Sequences</u><br> | ||
| + | Aer-EnvZ chimera:<br> | ||
| + | OmpR:<br> | ||
| + | Entire construct:</p> | ||
| + | <p><u>Upload aPe file here?<a name="_GoBack"></a></u></p> | ||
| + | </div> | ||
| + | <br clear="all"> | ||
| + | <div> </div> | ||
| + | <p> </p> | ||
| + | <h3> <span class="mw-headline" id="KanRDETAILS">KanR</span></h3> | ||
| + | <p>KanR provides <a href=" http://en.wikipedia.org/wiki/Kanamycin">kanamycin</a> resistance to colonies with successful transformations. <a name="_GoBack"></a></p> | ||
| + | <p> </p> | ||
| + | <h3> <span class="mw-headline" id="pBR322DETAILS">pBR322</span></h3> | ||
| + | <p>The <a href="http://openwetware.org/wiki/Escherichia_coli/Vectors ">pBR322</a> ori is a low copy origin of replication derived from <a name="_GoBack"></a></p> | ||
| + | <p> </p> | ||
| + | <h3> <span class="mw-headline" id="OmpRDETAILS">OmpR</span></h3> | ||
| + | <p>OmpR is the transcription factor regulated by EnvZ. It is found in the cytosol. OmpR is phosphorolized by the kinase EnvZ, making the two dependent on each other. OmpR’s N-terminus must be phosphorolized by EnvZ in order to allow transcription of certain transmembrane genes; the C-terminus moderates multimerization of OmpR, which in turn controls transcription of outer membrane proteins..<a name="_GoBack"></a></p> | ||
| + | <p> </p> | ||
| + | <h3> <span class="mw-headline" id="T7promoterDETAILS">T7 Promoter</span></h3> | ||
| + | <p>The T7 promoter is constitutively expressed by <a href="http://en.wikipedia.org/wiki/T7_RNA_polymerase">the phage T7 RNA polymerase</a>. The polymerase is orthogonal to the wildtype E. coli promoters.<a name="_GoBack"></a></p> | ||
| + | <p> </p> | ||
<p><s>This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go.This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. </s></p> | <p><s>This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go.This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. </s></p> | ||
<p><h1>Gel samples</h1></p> | <p><h1>Gel samples</h1></p> | ||
Revision as of 08:37, 5 July 2013
Aer
Aer
Aer is a sensory protein that measures oxygen levels within the cell, rather than externally. When low internal oxygen levels are sensed, it will trigger reactions such as movement towards quickly oxidizable substances such as glucose or ribose. It is located in the inner membrane of the cell. Aer also regulates the expression of many genes involved in anaerobic respiration. Aer is part of the PAS domain of sensing proteins.
EnvZ
Overview
EnvZ is the histidine kinase in the EnvZ-OmpR signal transduction system, located in the inner membrane. It contains two separate transmembrane regions. EnvZ autoposphorolates, then phosphorotales the transcription factor OmpR. EnvZ and OmpR are often seen as a single, polysistronic mRNA, although there is normally much less OmpR present in any given cell.
Aer is a sensory protein that measures oxygen levels within the cell, rather than externally. When low internal oxygen levels are sensed, it will trigger reactions such as movement towards quickly oxidizable substances such as glucose or ribose. It is located in the inner membrane of the cell. Aer also regulates the expression of many genes involved in anaerobic respiration. Aer is part of the PAS domain of sensing proteins.
We chose Aer because it is a more recently studied gene, so little is known about it. According to Nishiyama , Aer also has temperature sensing potential, so this could be another interesting aspect our project.
Our construct consists of an Aer-EnvZ chimera protein created view overlapping extension PCR (OEPCR), and a transcription factor, OmpR. EnvZ is a histidine kinase that communicates with OmpR.
Our platform for this construct:
Our construct:
Primers
EnvZ_R: aaaaaactcgagttaggatccTTACCCTTCTTTTGTCGTGCC*
OmpR_R: AAAAAACTCGAGAGGGGATCtcatgctttagagccgtccgg*
OmpR_F: aaaaaaCATATGCAAGAGACCTACAAGATTCTG
Aer_F: aaaaaagaattctaaagatctaaggagatataATGTCTTCTCATCCGTATGTC*
Aer-EnvZ_R: GCGCAAGTCGTGACTTACCCatttctgacactggacacctgg*
Aer-EnvZ_F: ccaggtgtccagtgtcagaaatGGGTAAGTCACGACTTGCGC*
*Lowercase text contains a restriction site
Construct Sequences
Aer-EnvZ chimera:
OmpR:
Entire construct:
KanR
KanR provides kanamycin resistance to colonies with successful transformations.
pBR322
The pBR322 ori is a low copy origin of replication derived from
OmpR
OmpR is the transcription factor regulated by EnvZ. It is found in the cytosol. OmpR is phosphorolized by the kinase EnvZ, making the two dependent on each other. OmpR’s N-terminus must be phosphorolized by EnvZ in order to allow transcription of certain transmembrane genes; the C-terminus moderates multimerization of OmpR, which in turn controls transcription of outer membrane proteins..
T7 Promoter
The T7 promoter is constitutively expressed by the phage T7 RNA polymerase. The polymerase is orthogonal to the wildtype E. coli promoters.
This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go.This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go.
Gel samples
This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go.This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go.
Flow sample
This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go.This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go. This is where the deatils of Aer will go.
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