Difference between revisions of "Team:Tacoma RAINmakers/Parts"

Line 115: Line 115:
 
</p>
 
</p>
 
<p class="listy">
 
<p class="listy">
−
     The Tacoma RAINMakers have built 2 biological arsenic circuits that use a combination of native E. coli and coral genes to detect arsenic ions in solution. The circuits vary in which reporter they produce, either chromoproteins spisPink (from S. pistillata) or amilCP (from A.millepora). Each circuit consists of a repressor, which is an arsenic regulator known as ArsR. This protein binds to a promoter containing an ArsR binding site (PArsR) and inhibits the transcription of the downstream reporter gene. Only in the presence of a transcription factor, which in our case is arsenic, does the reporter become derepressed and the reporter is expressed. This circuit allows us to control chromoprotein expression using a protein sensitive to and capable of binding with arsenic ions in solution.
+
     The Tacoma RAINMakers have built two biological arsenic circuits that use a combination of native E. coli and coral genes to detect arsenic ions in solution. The circuits vary in which reporter they produce, either the chromoprotein spisPink (from S. pistillata) or amilCP (from A.millepora). Each circuit contains an Arsenic regulator known as ArsR. The ArsR protein binds to a promoter with an ArsR binding site (PArsR) and inhibits the transcription of the downstream reporter gene. Only in the presence of a transcription factor, which in our case is arsenic, does the reporter become derepressed and the reporter is expressed. This circuit allows us to control chromoprotein expression using a protein sensitive to, and capable of binding with, arsenic ions.
 
<br><br>
 
<br><br>
−
    The most significant hazard in the RAINmaker project would be the use of chemicals containing arsenic.
+
</p>
−
Pure arsenic is not only toxic to humans, but it can also be highly toxic to aquatic life with lasting environmental and ecological effects. Sigma Aldrich warns that arsenic may also cause cancer. To work more safely, Tacoma RAINmakers used the sodium arsenate and sodium arsenite, which are the inorganic derivatives of arsenic that shows significantly less toxicity than the pure arsenic.
+
−
When using sodium arsenate/sodium arsenite, Tacoma RAINmakers are engaging in a series of stringent safety practices. First, safety glasses will always be used to protect eyes. For hand protection, our team will wear nitrile gloves that equal or exceed required layer thickness (0.11mm). Contaminated gloves will be disposed of with proper glove removal technique (without touching glove's outer surface). To protect the body, our team will wear lab coats during all arsenic-related experiments. RAINmakers have considered air-purifying respirators, but have deemed this precaution unnecessary with proper lab technique use of a biosafety cabinet. Arsenic waste solutions or powder will not be allowed to enter drains or any location outside the lab.</p>
+
  
  
Line 133: Line 131:
 
   <b>Part Type: Composite</b> <br>     
 
   <b>Part Type: Composite</b> <br>     
 
   <b>Description: </b>  
 
   <b>Description: </b>  
−
     pSB1C3/PcArsR_PArsR-spisPink is Tacoma RAINMaker’s arsenic detection circuit. It is a 2-part circuit that combines 2 series of promoter-gene sequences on one plasmid. The first part of the circuit, PcArsR, contains an E. coli-derived constitutive promoter (Pc), Ribosome Binding Site, Arsenic Regulator (ArsR), and Double Terminator. This sequence allows for unregulated expression of ArsR. The second part of the circuit contains an E. coli-derived Arsenic Regulator promoter (PArsR), Ribosome Binding Site, spisPink chromoprotein, and Double Terminator. The PArsR sequence has a binding site for ArsR, which is a repressor protein that blocks expression of the reporter. When Arsenic ions are present, the Arsenic Regulator proteins bind to those ions instead, allowing for spisPink chromoproteins to be produced.  
+
     pSB1C3/PcArsR_PArsR-spisPink is an arsenic detection circuit. It is a two-part circuit made up of a series of promoter-gene sequences. The first part of the circuit, PcArsR, contains an E. coli-derived constitutive promoter (Pc), a Ribosome Binding Site, Arsenic Regulator (ArsR), and a Double Terminator. This sequence creates unregulated expression of ArsR. The second part of the circuit contains an E. coli-derived Arsenic Regulator promoter (PArsR), a Ribosome Binding Site, the spisPink chromoprotein, and a Double Terminator. The PArsR sequence has a binding site for ArsR, which blocks expression of the reporter in the absence of Arsenic ions. When Arsenic ions are present the spisPink chromoproteins are produced.  
 
         <br>
 
         <br>
 
   <b>Website:</b> <a href = http://parts.igem.org/Part:BBa_K2673000>http://parts.igem.org/Part:BBa_K2673000</a> <br>         
 
   <b>Website:</b> <a href = http://parts.igem.org/Part:BBa_K2673000>http://parts.igem.org/Part:BBa_K2673000</a> <br>         
Line 146: Line 144:
 
   <b>Creator: Tacoma RAINmakers</b> <br>  
 
   <b>Creator: Tacoma RAINmakers</b> <br>  
 
   <b>Description: </b>  
 
   <b>Description: </b>  
−
     pSB1C3/PcArsR_PArsR-amilCP is Tacoma RAINMaker’s arsenic detection circuit. It is a 2-part circuit that combines 2 series of promoter-gene sequences on one plasmid. The first part of the circuit, PcArsR, contains an E. coli-derived constitutive promoter (Pc), Ribosome Binding Site, Arsenic Regulator (ArsR), and Double Terminator. This sequence allows for unregulated expression of ArsR. The second part of the circuit contains an E. coli-derived Arsenic Regulator promoter (PArsR), Ribosome Binding Site, amilCP chromoprotein, and Double Terminator. The PArsR sequence has a binding site for ArsR, which is a repressor protein that blocks expression of the reporter. When Arsenic ions are present, the Arsenic Regulator proteins bind to those ions instead, allowing for amilCP chromoproteins to be produced and fluoresce blue.
+
     pSB1C3/PcArsR_PArsR-amilCP is an arsenic detection circuit. It is a two-part circuit that combines two series of promoter-gene sequences on one plasmid. The first part of the circuit, PcArsR, contains an E. coli-derived constitutive promoter (Pc), a Ribosome Binding Site, Arsenic Regulator (ArsR), and a Double Terminator. This sequence allows for unregulated expression of ArsR. The second part of the circuit contains an E. coli-derived Arsenic Regulator promoter (PArsR), a Ribosome Binding Site, the amilCP chromoprotein, and a Double Terminator. The PArsR sequence has a binding site for ArsR which blocks expression of the reporter in the absence of Arsenic ions. When Arsenic ions are present amilCP chromoproteins are produced.
  
 
         <br>
 
         <br>
Line 160: Line 158:
 
   <b>Creator: Tacoma RAINmakers</b> <br>  
 
   <b>Creator: Tacoma RAINmakers</b> <br>  
 
   <b>Description: </b>  
 
   <b>Description: </b>  
−
   pSB1C3/PArsR-spisPink is used to test part of the complete 2-part arsenic circuit. The part contains an E. coli-derived Arsenic Regulator promoter (PArsR), Ribosome Binding Site, spisPink chromoprotein, and Double Terminator. The expression of the spisPink chromoprotein is regulated only if ArsR is present. In that case, ArsR will repress the promoter.
+
   pSB1C3/PArsR-spisPink is used to test part of the complete two-part arsenic sensing circuit. This part contains an E. coli-derived Arsenic Regulator promoter (PArsR), a Ribosome Binding Site, the spisPink chromoprotein, and a Double Terminator. The expression of the spisPink chromoprotein is regulated only if ArsR is present. In this plasmid the chromoprotein expression is unregulated.
  
  
Line 174: Line 172:
 
   <b>Creator: Tacoma RAINmakers</b> <br>  
 
   <b>Creator: Tacoma RAINmakers</b> <br>  
 
   <b>Description: </b>  
 
   <b>Description: </b>  
−
   The part contains the Ribosome Binding Site, PcArsR, and Double Terminator. This sequence allows for unregulated expression of ArsR, a regulatory protein.
+
   The part contains a Ribosome Binding Site, PcArsR, and a Double Terminator. This sequence creates  unregulated expression of ArsR. There is no reporter.
 
         <br>
 
         <br>
 
   <b>Website:</b> <a href =  http://parts.igem.org/Part:BBa_K2673003
 
   <b>Website:</b> <a href =  http://parts.igem.org/Part:BBa_K2673003
Line 188: Line 186:
 
   <b>Creator: Tacoma RAINmakers</b> <br>  
 
   <b>Creator: Tacoma RAINmakers</b> <br>  
 
   <b>Description: </b>  
 
   <b>Description: </b>  
−
   pSB1C3/PArsR-amilCP is used to test part of the complete 2-part arsenic circuit. The part contains an E. coli-derived Arsenic Regulator promoter (PArsR), Ribosome Binding Site, spisPink chromoprotein, and Double Terminator. The expression of the amilCP chromoprotein is regulated only if ArsR is present. In that case, ArsR will repress the promoter.  
+
   pSB1C3/PArsR-amilCP is used to test part of the complete two-part arsenic circuit. This part contains an E. coli-derived ArsR promoter (PArsR), a Ribosome Binding Site, the spisPink chromoprotein, and a Double Terminator. The expression of the amilCP chromoprotein is regulated only if ArsR is present. In this plasmid the chromoprotein expression is unregulated.
 +
 
  
 
         <br>
 
         <br>
Line 207: Line 206:
 
   <b>Creator: Tacoma RAINmakers</b> <br>  
 
   <b>Creator: Tacoma RAINmakers</b> <br>  
 
   <b>Description: </b>  
 
   <b>Description: </b>  
−
  AmilCP reporter driven by high expression T7 promoter.
+
To test the AmilCP promoter, we created a seperate circuit consisting of the AmilCP reporter an a high expression T7 promoter. This should produce unregulated chromoprotien, however in lab testing proved unsuccessful. We have speculated that this is due to a lack of sequence spacing around the promoter, but have yet to test this theory.
  
 
         <br>
 
         <br>

Revision as of 23:21, 15 October 2018

Team:TacomaRAINmakers/Notebook - 2017.igem.org

Team:ECUST/Lab/Notebook

Our Biobricks

Parts Overview


The Tacoma RAINMakers have built two biological arsenic circuits that use a combination of native E. coli and coral genes to detect arsenic ions in solution. The circuits vary in which reporter they produce, either the chromoprotein spisPink (from S. pistillata) or amilCP (from A.millepora). Each circuit contains an Arsenic regulator known as ArsR. The ArsR protein binds to a promoter with an ArsR binding site (PArsR) and inhibits the transcription of the downstream reporter gene. Only in the presence of a transcription factor, which in our case is arsenic, does the reporter become derepressed and the reporter is expressed. This circuit allows us to control chromoprotein expression using a protein sensitive to, and capable of binding with, arsenic ions.

Submitted Parts


BBa_K2673000 pSB1C3/PcArsR_PArsR-spisPink

Part Type: Composite
Description: pSB1C3/PcArsR_PArsR-spisPink is an arsenic detection circuit. It is a two-part circuit made up of a series of promoter-gene sequences. The first part of the circuit, PcArsR, contains an E. coli-derived constitutive promoter (Pc), a Ribosome Binding Site, Arsenic Regulator (ArsR), and a Double Terminator. This sequence creates unregulated expression of ArsR. The second part of the circuit contains an E. coli-derived Arsenic Regulator promoter (PArsR), a Ribosome Binding Site, the spisPink chromoprotein, and a Double Terminator. The PArsR sequence has a binding site for ArsR, which blocks expression of the reporter in the absence of Arsenic ions. When Arsenic ions are present the spisPink chromoproteins are produced.
Website: http://parts.igem.org/Part:BBa_K2673000

BBa_K2673001 pSB1C3/PcArsR_PArsR-amilCP

Part Type: Composite
Creator: Tacoma RAINmakers
Description: pSB1C3/PcArsR_PArsR-amilCP is an arsenic detection circuit. It is a two-part circuit that combines two series of promoter-gene sequences on one plasmid. The first part of the circuit, PcArsR, contains an E. coli-derived constitutive promoter (Pc), a Ribosome Binding Site, Arsenic Regulator (ArsR), and a Double Terminator. This sequence allows for unregulated expression of ArsR. The second part of the circuit contains an E. coli-derived Arsenic Regulator promoter (PArsR), a Ribosome Binding Site, the amilCP chromoprotein, and a Double Terminator. The PArsR sequence has a binding site for ArsR which blocks expression of the reporter in the absence of Arsenic ions. When Arsenic ions are present amilCP chromoproteins are produced.
Website: http://parts.igem.org/Part:BBa_K2673001

BBa_K2673002 pSB1C3/PArsR-spisPink

Part Type: Composite
Creator: Tacoma RAINmakers
Description: pSB1C3/PArsR-spisPink is used to test part of the complete two-part arsenic sensing circuit. This part contains an E. coli-derived Arsenic Regulator promoter (PArsR), a Ribosome Binding Site, the spisPink chromoprotein, and a Double Terminator. The expression of the spisPink chromoprotein is regulated only if ArsR is present. In this plasmid the chromoprotein expression is unregulated.
Website: http://parts.igem.org/Part:BBa_K2673002

BBa_K2673003 pSB1C3/PcArsR

Part Type: Composite
Creator: Tacoma RAINmakers
Description: The part contains a Ribosome Binding Site, PcArsR, and a Double Terminator. This sequence creates unregulated expression of ArsR. There is no reporter.
Website: http://parts.igem.org/Part:BBa_K2673003

BBa_K2673004 pSB1C3/PArsR-amilCP

Part Type: Composite
Creator: Tacoma RAINmakers
Description: pSB1C3/PArsR-amilCP is used to test part of the complete two-part arsenic circuit. This part contains an E. coli-derived ArsR promoter (PArsR), a Ribosome Binding Site, the spisPink chromoprotein, and a Double Terminator. The expression of the amilCP chromoprotein is regulated only if ArsR is present. In this plasmid the chromoprotein expression is unregulated.
Website: http://parts.igem.org/Part:BBa_K2673004

BBa_K2673005 pSB1C3/T7-amilCP

Part Type: Composite
Creator: Tacoma RAINmakers
Description: To test the AmilCP promoter, we created a seperate circuit consisting of the AmilCP reporter an a high expression T7 promoter. This should produce unregulated chromoprotien, however in lab testing proved unsuccessful. We have speculated that this is due to a lack of sequence spacing around the promoter, but have yet to test this theory.
Website: http://parts.igem.org/Part:BBa_K2673005