Difference between revisions of "Part:BBa K3657013"

Latest revision as of 03:37, 28 October 2020

RNA binding Pentatricopeptide Repeat Protein - Motif GUAGAG

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

It is an OFF target PPR Protein for plasmides, containing the RBS BBa_B0032 because it binds to the RNA sequence GUAGAG, which is not found in the RBS. It is an ON target PPR Protein the broccoli aptamer because it binds to the RNA sequence ACACAG, which can be find in the loop or stem region of the broccoli aptamer.
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_K3657013, an assay was conducted, measuring the expression of superfoldGFP under the promotor BBa_B0032. Since this PPR Protein can not bind to the RBS, it isn't hindering translation of the mRNA. The expression of GFP should therefore not be reduced compared to the sample containing the PPR Protein BBa_K3657012, which is 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]

@@ Line 11: / Line 11: @@
 Cytosine:<a href="https://parts.igem.org/Part:BBa_K3657009">BBa_K3657009</a><p>
+It is an OFF 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 GUAGAG, which is not found in the RBS.
+It is an ON target PPR Protein the broccoli aptamer because it binds to the RNA sequence ACACAG, which can be find in the loop or stem region of the broccoli aptamer.
+<br>
+For more information, visit <a href="https://2020.igem.org/Team:Heidelberg/Pumilio_PPR#pumby">iGEM Heidelberg 2020</a></p>
 </html>
@@ Line 17: / Line 21: @@
 PPRs are originally found in <i> Arabidopsis thaliana </i> and can bind RNA. In chloroplasts, they take part in many post-translational regulation processes.
+==Characterisation==
+<html>
+In combination with part <a href="https://parts.igem.org/Part:BBa_K3657013">BBa_K3657013</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 not bind to the RBS, it isn't hindering translation of the mRNA. The expression of GFP should therefore not be reduced compared to the sample containing the PPR Protein <a href="https://parts.igem.org/Part:BBa_K3657012">BBa_K3657012</a>, which is 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:
+</html>
+<html>
+<center>
+<img style="width:75%" src="https://2020.igem.org/wiki/images/4/49/T--Heidelberg--measurementpprs.png">
+</center>
+</html>
+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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