Difference between revisions of "Part:BBa K3011007"

 
 
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__NOTOC__
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<partinfo>BBa_K3011007 short</partinfo>
  
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NsrR (BBa_K1682011) is a Nitric Oxide (NO) sensitive transcription repressor. This construct produces NsrR constitutively; in absence of NO, NsrR binds to the NsrR binding site present on the PyeaR promoter (BBa_K216005) and repress the transcription of mRFP (BBa_E1010). In presence of NO, NsrR binds to NO due to which NsrR can’t bind to the DNA (thus can’t repress gene expression) and mRFP will produce.
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NsrR contains a Helix Turn Helix (HTH) motif and Fe-S cluster ([2Fe-2S] or [4Fe-4S]). In absence of NO, NsrR binds to a particular DNA sequence by using HTH motif (as a homodimer) and repress gene expression. In presence of NO, Fe-S cluster reacts with NO to form dinitrosyl iron complex, due to the formation of this complex NsrR can’t bind to that particular sequence anymore. Depending upon the sequence of NsrR binding site, the number of NO molecules (per cluster) which is required for abolishing DNA binding vary (2-10 NO / cluster)
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<b> Characterization </b>
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This new biobrick part can be activated in a certain concentration (10^-4 M to 10^-5 M) of Nitric Oxide in the media.
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                      https://2019.igem.org/wiki/images/d/d2/T--iiser_kolkata--colony_pcr_ga.jpeg
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<b>colony PCR was done to confirm the presence of correct transformants.</b>
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For colony PCR we used the vector specific VF2 and VR that when simulated on SnapGene showed a amplicon of 1730 bp. This was confirmed by the PCR products run on the gel. For the colonies with positive amplicon we grew them in liquid media until it reached an OD of around 0.6 and then it was induced by Sodium Nitroprusside solution which releases NO in aquatic media.
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https://2019.igem.org/wiki/images/e/ec/T--iiser_kolkata--SNP_red_colony_induc.jpeg
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<b>We induced the bacterial culture with 10^-4 M Nitric Oxide concentration and the induced colonies were producing mRFP after 6 hours.</b>
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We got our colony on the very last day so we couldn't manage to get a fluorescence data for the exact quantification of mRFP production but qualitatively it can be said that there is clear induction of Pyear promoter in the presence of Nitric Oxide in the medium. The concentration was determined from the characterization of part  BBa_K381001 with Pyear promoter controlling GFP production. The details of the same can be found on this link (https://2019.igem.org/Team:IISER_Kolkata/Contribution).
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3011007 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K3011007 parameters</partinfo>
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Latest revision as of 02:24, 22 October 2019

Nitric Oxide sensor

NsrR (BBa_K1682011) is a Nitric Oxide (NO) sensitive transcription repressor. This construct produces NsrR constitutively; in absence of NO, NsrR binds to the NsrR binding site present on the PyeaR promoter (BBa_K216005) and repress the transcription of mRFP (BBa_E1010). In presence of NO, NsrR binds to NO due to which NsrR can’t bind to the DNA (thus can’t repress gene expression) and mRFP will produce.

NsrR contains a Helix Turn Helix (HTH) motif and Fe-S cluster ([2Fe-2S] or [4Fe-4S]). In absence of NO, NsrR binds to a particular DNA sequence by using HTH motif (as a homodimer) and repress gene expression. In presence of NO, Fe-S cluster reacts with NO to form dinitrosyl iron complex, due to the formation of this complex NsrR can’t bind to that particular sequence anymore. Depending upon the sequence of NsrR binding site, the number of NO molecules (per cluster) which is required for abolishing DNA binding vary (2-10 NO / cluster)

Characterization This new biobrick part can be activated in a certain concentration (10^-4 M to 10^-5 M) of Nitric Oxide in the media. 

                      T--iiser_kolkata--colony_pcr_ga.jpeg

colony PCR was done to confirm the presence of correct transformants.

For colony PCR we used the vector specific VF2 and VR that when simulated on SnapGene showed a amplicon of 1730 bp. This was confirmed by the PCR products run on the gel. For the colonies with positive amplicon we grew them in liquid media until it reached an OD of around 0.6 and then it was induced by Sodium Nitroprusside solution which releases NO in aquatic media.


T--iiser_kolkata--SNP_red_colony_induc.jpeg We induced the bacterial culture with 10^-4 M Nitric Oxide concentration and the induced colonies were producing mRFP after 6 hours.

We got our colony on the very last day so we couldn't manage to get a fluorescence data for the exact quantification of mRFP production but qualitatively it can be said that there is clear induction of Pyear promoter in the presence of Nitric Oxide in the medium. The concentration was determined from the characterization of part BBa_K381001 with Pyear promoter controlling GFP production. The details of the same can be found on this link (https://2019.igem.org/Team:IISER_Kolkata/Contribution).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 202
    Illegal AgeI site found at 1222
    Illegal AgeI site found at 1334
  • 1000
    COMPATIBLE WITH RFC[1000]