Difference between revisions of "Part:BBa K1602046"
 
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        <h1>RRK3</h1>
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                <b>RRK3</b> is a composite of the araCpBad promotor (BBa_K808000) which masks the key3(BBa_J01086).
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Composite part consisting of an araC-regulated pBAD-promoter (<html><a href="/Part:BBa_K808000">BBa_K808000</a></html>), Key3 (<html><a href="/Part:BBa_J01086">BBa_J01086</a></html>) and a terminator (<html><a href="/Part:BBa_B0015">BBa_B0015</a></html>).
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RRK3 belongs to a two-part riboregulator-system for <i>E.coli</i> for posttransciptional regulation of gene expression. Upon transcription the sequence of <html><a href="/Part:BBa_J01086">Key3</a></html>(taRNA) forms a RNA-RNA-complex with corresponding cis-repressing sequences (crRNA). This leads to a helix shift and the release of the formerly masked ribosome binding site (RBS) enabling the expression of the regulated gene of interest (GOI). The corresponding crRNAs are <html><a href="/Part:BBa_K1602044">RRL3H</a></html> and <html><a href="/Part:BBa_K1602045">RRL3G</a></html>.
  
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<figcaption><b>Figure 1:</b> Interaction of taRNA and crRNA leads to gene expression.</figcaption>
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Because of the <html><a href="/Part:BBa_K808000">araC-regulated pBAD-promoter</a></html> production of the Lock3-sequence is induced by the addition of arabniose. In the presence of glucose araCpBAD shows a very low basal expression level. In combination with the corresponding crRNA-parts (<html><a href="/Part:BBa_K1602044">RRL3H</a></html> and <html><a href="/Part:BBa_K1602045">RRL3G</a></html>) this results in an induceable riboregulator-system which represses gene expression in the presence of glucose but is inducable through the addition of arabinose.
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===Functional Parameters===
 
===Functional Parameters===
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In order to assemble the final riboregulator the parts RRL3G (<html><a href="/Part:BBa_K1602045">BBa_K1602045</a></html>) and RRK3 (<html><a href="/Part:BBa_K1602046">BBa_K1602046</a></html>) on two seperate plasmids (pSB1C3 and pSB1A2) were co-transformed into <i>E.coli</i>(Top10). Positive transformants were selected by using two antibiotics, Chloramphenicol and Ampicillin, and verified via colony-PCR.
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As controls served a culture of TOP10 without plasmid, one transformed with araCpBad-GFP (<html><a href="/Part:BBa_K1602055">BBa_K1602055</a></html>) as positive control and one transformed only with the cis-repressed part of the riboregulator (<html><a href="/Part:BBa_K1602045">RRL3G - BBa_K1602045</a></html>) as negative control. All four cultures were grown in LB-medium with the respective antibiotics containing 20mM glucose at 37°C over night. Afterwards 10µl of each culture were inoculated in two seperate flasks of LB-medium (with the respective antibiotics), one containing 20mM Glucose, the other one 2mM arabinose. After 16 hours of incubation at 37°C 1 ml of each culture was pelleted by centrifugation and resuspended in PBS for subsequent FACS-measurements.
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<figcaption><b>Figure 2:</b> Results of the FACS-measurements. A:negative control (TOP10) B:positive control (araC-pBAD-GFP) C: negative control (RRL3G) D:fully assembled riboregulator (RRK3/RRL3G)</figcaption>
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The positive control (Fig.2 B) showed a significant difference in the detected fluorescence-levels between the culture grown with glucose and the culture grown with arabinose indicating that the addition of 20mM glucose to the medium is sufficient to repress GFP-expression through the araC-regulated pBAD-promoter. Surprisingly we were not able to detect the same difference in the cultures containing the assembled riboregulator (Fig.2 D). The measured fluorescence for the induced culture grown with arabinose was the same as for the culture grown with glucose. Furthermore was the detected GFP-Signal very simmilar to the results of the negativ controls (Fig.2 A+C) what leads to the conclusion that no GFP was expressed in the culture containing the riboregulator at all.
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We have to assume that the interaction between the two parts of the riboregulator does not happen as anticipated, leaving the riboregulator-system constantly "locked" and preventing GFP expression, even after induction.
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One possible reason for the malfunction of the riboregulator could be the fact that both parts were located on seperate plasmids which were co-transformed into the cells. It is possible that this results in an unfavorable situation for the bacteria to produce enough of both parts necessary for the riboregulator to work.
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To further investigate this hypothesis we cloned both parts of the riboregulator next to each other on one plasmid resulting in the BioBrick RRK3-RRL3G (<html><a href="/Part:BBa_K1602047">BBa_K1602047</a></html>) but we were not able to repeat the experiment so far due to time constraints.
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===References===
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1602046 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K1602046 parameters</partinfo>
 
<partinfo>BBa_K1602046 parameters</partinfo>
 
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Latest revision as of 23:15, 25 September 2015

RRK3

Composite part consisting of an araC-regulated pBAD-promoter (BBa_K808000), Key3 (BBa_J01086) and a terminator (BBa_B0015). RRK3 belongs to a two-part riboregulator-system for E.coli for posttransciptional regulation of gene expression. Upon transcription the sequence of Key3(taRNA) forms a RNA-RNA-complex with corresponding cis-repressing sequences (crRNA). This leads to a helix shift and the release of the formerly masked ribosome binding site (RBS) enabling the expression of the regulated gene of interest (GOI). The corresponding crRNAs are RRL3H and RRL3G.

Figure 1: Interaction of taRNA and crRNA leads to gene expression.

Because of the araC-regulated pBAD-promoter production of the Lock3-sequence is induced by the addition of arabniose. In the presence of glucose araCpBAD shows a very low basal expression level. In combination with the corresponding crRNA-parts (RRL3H and RRL3G) this results in an induceable riboregulator-system which represses gene expression in the presence of glucose but is inducable through the addition of arabinose.


Functional Parameters

In order to assemble the final riboregulator the parts RRL3G (BBa_K1602045) and RRK3 (BBa_K1602046) on two seperate plasmids (pSB1C3 and pSB1A2) were co-transformed into E.coli(Top10). Positive transformants were selected by using two antibiotics, Chloramphenicol and Ampicillin, and verified via colony-PCR.

As controls served a culture of TOP10 without plasmid, one transformed with araCpBad-GFP (BBa_K1602055) as positive control and one transformed only with the cis-repressed part of the riboregulator (RRL3G - BBa_K1602045) as negative control. All four cultures were grown in LB-medium with the respective antibiotics containing 20mM glucose at 37°C over night. Afterwards 10µl of each culture were inoculated in two seperate flasks of LB-medium (with the respective antibiotics), one containing 20mM Glucose, the other one 2mM arabinose. After 16 hours of incubation at 37°C 1 ml of each culture was pelleted by centrifugation and resuspended in PBS for subsequent FACS-measurements.

Figure 2: Results of the FACS-measurements. A:negative control (TOP10) B:positive control (araC-pBAD-GFP) C: negative control (RRL3G) D:fully assembled riboregulator (RRK3/RRL3G)

The positive control (Fig.2 B) showed a significant difference in the detected fluorescence-levels between the culture grown with glucose and the culture grown with arabinose indicating that the addition of 20mM glucose to the medium is sufficient to repress GFP-expression through the araC-regulated pBAD-promoter. Surprisingly we were not able to detect the same difference in the cultures containing the assembled riboregulator (Fig.2 D). The measured fluorescence for the induced culture grown with arabinose was the same as for the culture grown with glucose. Furthermore was the detected GFP-Signal very simmilar to the results of the negativ controls (Fig.2 A+C) what leads to the conclusion that no GFP was expressed in the culture containing the riboregulator at all.

We have to assume that the interaction between the two parts of the riboregulator does not happen as anticipated, leaving the riboregulator-system constantly "locked" and preventing GFP expression, even after induction.

One possible reason for the malfunction of the riboregulator could be the fact that both parts were located on seperate plasmids which were co-transformed into the cells. It is possible that this results in an unfavorable situation for the bacteria to produce enough of both parts necessary for the riboregulator to work.

To further investigate this hypothesis we cloned both parts of the riboregulator next to each other on one plasmid resulting in the BioBrick RRK3-RRL3G (BBa_K1602047) but we were not able to repeat the experiment so far due to time constraints.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1215
    Illegal SapI site found at 961