Team:UPO-Sevilla/Project/Improving Flip Flop/Data Page

From 2011.igem.org

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                             <h1>Data Page</h1>     
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"color" : "black",
"color" : "black",
"position" : "T",
"position" : "T",
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"text" : "When we give a heat shock to our bacteria (42 ºC) cI promoter (PcI) is activated, expressing LacI::Das+4 and GFP::Das+4 proteins. Now we are in State 1. ",
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"text" : "When we give a heat shock to our bacteria (42 ºC) cI promoter (PcI) is activated, expressing LacI::Das+4 and GFP::Das+4 proteins. Now we are in Green State. ",
"time" : 5000
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},
},
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"bgcolor" : "white",
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"text" : "LacI::Das+4 is Lac promoter repressor. This protein represses PLac and, therefore, State 2. This will assure de maintenance of State 1 over State 2 in absence of State 2 stimulus.",
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"text" : "LacI::Das+4 is Lac promoter repressor. This protein represses PLac and, therefore, Red State. This will assure de maintenance of Green State over Red State in absence of Red State stimulus.",
"position" : "T",
"position" : "T",
"time" : 5000
"time" : 5000
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"bgcolor" : "white",
"bgcolor" : "white",
"color" : "black",
"color" : "black",
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"text" : "GFP::Das+4 is State 1 reporter, which makes our bacteria glow in green when exposed to UV.",
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"text" : "GFP::Das+4 is Green State reporter, which makes our bacteria glow in green when exposed to UV.",
"position" : "T",
"position" : "T",
"time" : 5000
"time" : 5000
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"bgcolor" : "white",
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"text" : "Additionally, RybB asRNA is also regulated by PcI promoter so that it is also expressed when we give the heat shock. RybB bind State 2 OmpN::SspB’s and OmpN::RFP’s mRNA as they have its binding sequence fused (OmpN protein start sequence). This union prevents these mRNA’s translation, so that we are also eliminating State 2 reporter and SspB adaptor when expressing State 1.",
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"text" : "Additionally, RybB asRNA is also regulated by PcI promoter so that it is also expressed when we give the heat shock. RybB bind Red State OmpN::SspB’s and OmpN::RFP’s mRNA as they have its binding sequence fused (OmpN protein start sequence). This union prevents these mRNA’s translation, so that we are also eliminating Red State reporter and SspB adaptor when expressing Green State.",
"position" : "T",
"position" : "T",
"time" : 5000
"time" : 5000
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"bgcolor" : "white",
"bgcolor" : "white",
"color" : "black",
"color" : "black",
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"text" : "When we give an IPTG pulse to our bacteria Lac promoter (PLac) is activated, expressing cI (temperature-sensitive), OmpN::SspB and OmpN::RFP proteins. Now we are in State 2. ",
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"text" : "When we give an IPTG pulse to our bacteria Lac promoter (PLac) is activated, expressing cI (temperature-sensitive), OmpN::SspB and OmpN::RFP proteins. Now we are in Red State. ",
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"position" : "T",
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"position" : "B",
"time" : 5000
"time" : 5000
},
},
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"bgcolor" : "white",
"bgcolor" : "white",
"color" : "black",
"color" : "black",
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"text" : "cI is cI promoter repressor. This protein represses PcI and, therefore, State 1. This will assure de maintenance of State 2 over State 1 in absence of State 1 stimulus. As this cI is temperature sensitive it is degraded when we give our bacteria the heat shock necessary for State 1 activation.",
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"text" : "cI is cI promoter repressor. This protein represses PcI and, therefore, Green State. This will assure de maintenance of Red State over Green State in absence of State Green stimulus. As this cI is temperature sensitive it is degraded when we give our bacteria the heat shock necessary for Green State activation.",
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"position" : "T",
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"position" : "B",
"time" : 5000
"time" : 5000
},
},
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"bgcolor" : "white",
"bgcolor" : "white",
"color" : "black",
"color" : "black",
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"text" : "OmpN::RFP is State 2 reporter, which makes our bacteria glow in red when exposed to UV.",
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"text" : "OmpN::RFP is Red State reporter, which makes our bacteria glow in red when exposed to UV.",
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"position" : "T",
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"position" : "B",
"time" : 5000
"time" : 5000
},
},
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"bgcolor" : "white",
"bgcolor" : "white",
"color" : "black",
"color" : "black",
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"text" : "SspB is the adaptor protein that allows the ClpXP protease to degrade DAS+4 tagged proteins, in this case LacI::DAS+4  and GFP::DAS+4. Therefore, State 2 expression also eliminates State 1 reporter and State 2 repressor. ",
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"text" : "SspB is the adaptor protein that allows the ClpXP protease to degrade DAS+4 tagged proteins, in this case LacI::DAS+4  and GFP::DAS+4. Therefore, Red State expression also eliminates Green State reporter and Red State repressor. ",
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"position" : "T",
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"position" : "B",
"time" : 5000
"time" : 5000
},
},
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"color" : "black",
"color" : "black",
"text" : "This State won’t change again until we give our bacterias the heat shock.",
"text" : "This State won’t change again until we give our bacterias the heat shock.",
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"position" : "T",
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"position" : "B",
"time" : 5000
"time" : 5000
},
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         </script>   
         </script>   
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<br>
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                          <p>The <strong>Improved Flip Flop</strong> (Part: <a href="http://partsregistry.org/Part:BBa_K510019" target="_blank">BBa_K510019</a> and Part: <a href="http://partsregistry.org/Part:BBa_K510036" target="_blank">BBa_K510036</a>) is based on the basic flip flop (Part: <a href="http://partsregistry.org/Part:BBa_K177038" target="_blank">BBa_K177038</a>) and was built with devices which are in the Registry of Standard Biological Parts. As for the different sequences used in our construction, here it is a brief description of each one:</p>
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<br>
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<ol>
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<li><p> <strong>cI promoter </strong>(Part: <a href="http://partsregistry.org/Part:BBa_R0051" target="_blank">BBa_R0051</a>): The cI regulated promoter is based on the pR promoter from bacteriophage lambda. The promoter has two DNA binding sites for lambda cI repressor BBa_C0051. cI binding results in repression of transcription. The specific sequence used here is based on the cI repressible promoter used in the Elowitz repressilator (and references therein).</p></li>
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<li><p><strong>Lac promoter </strong>(Part: <a href="http://partsregistry.org/Part:BBa_R0010" target="_blank">BBa_R0010</a>): This part is an inverting regulator sensitive to LacI and CAP. It contains two protein binding sites. The first binds CAP protein, which is generally present in E.coli and is associated with cell health and availability of glucose. The second one binds LacI protein.</p> </li>
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<li><p><strong>RBS.3 medium </strong> (Part: <a href="http://partsregistry.org/Part:BBa_B0032" target="_blank">BBa_B0032</a>):This part is a weak RBS based on Ron Weiss thesis.</p> </li>
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<li><p><strong>LacI:</strong> Our LacI was taken from E. coli strain K-12 substrain MG1655 chromosome genome and it has a total of 1083 bp.</p></li>
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<li><p><strong>GFP </strong>(Part: <a href="http://partsregistry.org/Part:BBa_E0040" target="_blank">BBa_E0040</a>): Green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP.</p></li>
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<li><p><strong>DAS+4:</strong> Short tag at the end of the proteins. For more information see Figure 2 of Proteolysis section.</p></li>
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<li><p><strong>Temperature sensitive cI </strong>(Part: <a href="http://partsregistry.org/Part:BBa_K177050" target="_blank">BBa_K177050</a>): Thermosensitive version of the lambda phage repressor. Functions in 30°C and it's inactivated in 42°C</p></li>
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<li><p><strong>SspB:</strong> Taken from <a href="http://www.ncbi.nlm.nih.gov/pubmed/16762842" target="_blank">McGinness et al. (2006)</a>. For more information, see Proteolysis section. The OmpN::SspB fusion is detailed in Figure 5 of the asRNA section.</p> </li>
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<li><p><strong>RFP </strong>(Part: <a href="http://partsregistry.org/Part:BBa_E1010" target="_blank">BBa_E1010</a>): Highly engineered mutant of red fluorescent protein from Discosoma striata (coral). Monomeric RFP with its excitation peak at  584 nm and its emission peak at 607 nm. The OmpN::RFP fusion is detailed in Figure 5 of the asRNA section.</p></li>
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<li><p><strong>Double terminator </strong>(Part <a href="http://partsregistry.org/Part:BBa_B0015" target="_blank">BBa_B0015</a>): Double terminator consisting of <a href="http://partsregistry.org/Part:BBa_B0010" target="_blank">BBa_B0010</a> and <a href="http://partsregistry.org/Part:BBa_B0012" target="_blank">BBa_B0012</a>.This is the most commonly used terminator and it seems to be reliable.</p></li>
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<li><p><strong>RybB asRNA:</strong> Taken from <a href="http://www.ncbi.nlm.nih.gov/pubmed/19111662"_blank">Bouvier et al.(2008)</a>. For further information see Figure 2 of the asRNA section.</p></li>
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Latest revision as of 22:14, 27 October 2011

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Data Page


The Improved Flip Flop (Part: BBa_K510019 and Part: BBa_K510036) is based on the basic flip flop (Part: BBa_K177038) and was built with devices which are in the Registry of Standard Biological Parts. As for the different sequences used in our construction, here it is a brief description of each one:


  1. cI promoter (Part: BBa_R0051): The cI regulated promoter is based on the pR promoter from bacteriophage lambda. The promoter has two DNA binding sites for lambda cI repressor BBa_C0051. cI binding results in repression of transcription. The specific sequence used here is based on the cI repressible promoter used in the Elowitz repressilator (and references therein).

  2. Lac promoter (Part: BBa_R0010): This part is an inverting regulator sensitive to LacI and CAP. It contains two protein binding sites. The first binds CAP protein, which is generally present in E.coli and is associated with cell health and availability of glucose. The second one binds LacI protein.

  3. RBS.3 medium (Part: BBa_B0032):This part is a weak RBS based on Ron Weiss thesis.

  4. LacI: Our LacI was taken from E. coli strain K-12 substrain MG1655 chromosome genome and it has a total of 1083 bp.

  5. GFP (Part: BBa_E0040): Green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP.

  6. DAS+4: Short tag at the end of the proteins. For more information see Figure 2 of Proteolysis section.

  7. Temperature sensitive cI (Part: BBa_K177050): Thermosensitive version of the lambda phage repressor. Functions in 30°C and it's inactivated in 42°C

  8. SspB: Taken from McGinness et al. (2006). For more information, see Proteolysis section. The OmpN::SspB fusion is detailed in Figure 5 of the asRNA section.

  9. RFP (Part: BBa_E1010): Highly engineered mutant of red fluorescent protein from Discosoma striata (coral). Monomeric RFP with its excitation peak at 584 nm and its emission peak at 607 nm. The OmpN::RFP fusion is detailed in Figure 5 of the asRNA section.

  10. Double terminator (Part BBa_B0015): Double terminator consisting of BBa_B0010 and BBa_B0012.This is the most commonly used terminator and it seems to be reliable.

  11. RybB asRNA: Taken from Bouvier et al.(2008). For further information see Figure 2 of the asRNA section.

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