Difference between revisions of "Part:BBa K3657012"
 
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Cytosine:<a href="https://parts.igem.org/Part:BBa_K3657009">BBa_K3657009</a><p>
 
Cytosine:<a href="https://parts.igem.org/Part:BBa_K3657009">BBa_K3657009</a><p>
  
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It is an ON target PPR Protein for  binding the RNA sequence ACACAG which cannot be found in the broccoli aptamers.
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It is an ON target PPR Protein for plasmides, containing the RBS <a href="https://parts.igem.org/Part:BBa_B0032">BBa_B0032</a> because it binds to the RNA sequence ACACAG, which is part of the RBS.
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It is an OFF target PPR Protein for the broccoli aptamer because it binds to the RNA sequence ACACAG,  which cannot be found in the broccoli aptamers.
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For more information, visit <a href="https://2020.igem.org/Team:Heidelberg/Pumilio_PPR#pumby">iGEM Heidelberg 2020</a></p>
 
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PPRs are originally found in <i> Arabidopsis thaliana </i> and can bind RNA. In chloroplasts, they take part in many post-translational regulation processes.
 
PPRs are originally found in <i> Arabidopsis thaliana </i> and can bind RNA. In chloroplasts, they take part in many post-translational regulation processes.
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==Characterisation==
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In combination with part <a href="https://parts.igem.org/Part:BBa_K3657012">BBa_K3657012</a>, an assay was conducted, measuring the expression of superfoldGFP under the promotor <a href="https://parts.igem.org/Part:BBa_B0032">BBa_B0032</a>. Since this PPR Protein can bind to the RBS, thus hindering translation of the mRNA, the expression of GFP should be reduced compared to the sample containing the PPR Protein<a href="https://parts.igem.org/Part:BBa_K3657012">BBa_K3657012</a>, which is not able to bind to the RBS. Both PPR proteins were transformed together with sfGFP and the flourescence was measured in comparison to only sfGFP(positive control) and no sfGFP (negative control). The results were as follows:
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<img style="width:75%" src="https://2020.igem.org/wiki/images/4/49/T--Heidelberg--measurementpprs.png">
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The boxplot suggests that there is no significant difference in sfGFP expression between the positiveprobe containing solely sfGFP and the sample containing the ON target PPR. The negative control expressing an OFF target PPRs exhibited even a lower fluorescence. Although these results suggest that there does not seem to be an inhibition originating from the PPR proteins, we would not already call them off. The Anderson promoter BBa_J23100 expresses proteins 6.4 times stronger than the BBa_J23115 expressing the PPR proteins. Additionally, the PPR proteins were cloned into a plasmid containing the p15A ORI, which has a lower replication number than the ColE1 ORI that the sfGFP Reporter plasmid contains.
  
 
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<partinfo>BBa_K3657012 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3657012 SequenceAndFeatures</partinfo>
  
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==Characterisation==
 
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In combination with part BBa K3657012, an assay was conducted, messuaring
 
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 03:40, 28 October 2020


RNA binding Pentatricopeptide Repeat Protein - Motif ACACAG


This Part is an example of a PPR Protein, assembled with the PPR Domains that are specific for the four RNA bases.

Adenine:BBa_K3657006

Guanine:BBa_K3657007

Uracil:BBa_K3657008

Cytosine:BBa_K3657009

It is an ON target PPR Protein for plasmides, containing the RBS BBa_B0032 because it binds to the RNA sequence ACACAG, which is part of the RBS. It is an OFF target PPR Protein for the broccoli aptamer because it binds to the RNA sequence ACACAG, which cannot be found in the broccoli aptamers.
For more information, visit iGEM Heidelberg 2020

Usage and Biology

PPRs are originally found in Arabidopsis thaliana and can bind RNA. In chloroplasts, they take part in many post-translational regulation processes.


Characterisation

In combination with part BBa_K3657012, an assay was conducted, measuring the expression of superfoldGFP under the promotor BBa_B0032. Since this PPR Protein can bind to the RBS, thus hindering translation of the mRNA, the expression of GFP should be reduced compared to the sample containing the PPR ProteinBBa_K3657012, which is not able to bind to the RBS. Both PPR proteins were transformed together with sfGFP and the flourescence was measured in comparison to only sfGFP(positive control) and no sfGFP (negative control). The results were as follows:

The boxplot suggests that there is no significant difference in sfGFP expression between the positiveprobe containing solely sfGFP and the sample containing the ON target PPR. The negative control expressing an OFF target PPRs exhibited even a lower fluorescence. Although these results suggest that there does not seem to be an inhibition originating from the PPR proteins, we would not already call them off. The Anderson promoter BBa_J23100 expresses proteins 6.4 times stronger than the BBa_J23115 expressing the PPR proteins. Additionally, the PPR proteins were cloned into a plasmid containing the p15A ORI, which has a lower replication number than the ColE1 ORI that the sfGFP Reporter plasmid contains.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]