Difference between revisions of "Part:BBa K1602045"

 
 
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<partinfo>BBa_K1602045 short</partinfo>
 
<partinfo>BBa_K1602045 short</partinfo>
  
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Composite part consisting of a constitutive promoter (<html><a href="/Part:BBa_J23100">BBa_J23100</a></html>), Lock3 (<html><a href="/Part:BBa_J01080">BBa_J01080</a></html>) and GFP (<html><a href="/Part:BBa_E0040">BBa_E0040</a></html>). It belongs to a two-part riboregulator-system for posttransciptional regulation of GFP-expression. Upon transcription the sequence of <html><a href="/Part:BBa_J01080">Lock3</a></html> forms a hairpin-secondary-structure masking the ribosome binding site (RBS) and therefore prevents translation of the following GFP-sequence.
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If the corresponding trans-activating RNA-sequence (taRNA) (<html><a href="/Part:BBa_J01086">Key3-BBa_J01086</a></html>) is present the two sequences form a RNA-RNA-complex. This leads to a helix shift and the release of the RBS enabling the expression of GFP as the gene of interest (GOI).
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<figure >
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<img width=30%; src="https://static.igem.org/mediawiki/parts/3/3d/Schema_GOI.png">
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<figcaption><b>Figure 1:</b> Interaction of taRNA and crRNA leads to expression of GFP.</figcaption>
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===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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<figure >
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<img width=50%; src="https://static.igem.org/mediawiki/parts/8/86/FACS-registry-RRL3G_final.png">
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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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===Functional Parameters===
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<partinfo>BBa_K1602045 parameters</partinfo>
 
<partinfo>BBa_K1602045 parameters</partinfo>
 
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Latest revision as of 09:22, 25 September 2015

RRL3G

Composite part consisting of a constitutive promoter (BBa_J23100), Lock3 (BBa_J01080) and GFP (BBa_E0040). It belongs to a two-part riboregulator-system for posttransciptional regulation of GFP-expression. Upon transcription the sequence of Lock3 forms a hairpin-secondary-structure masking the ribosome binding site (RBS) and therefore prevents translation of the following GFP-sequence. If the corresponding trans-activating RNA-sequence (taRNA) (Key3-BBa_J01086) is present the two sequences form a RNA-RNA-complex. This leads to a helix shift and the release of the RBS enabling the expression of GFP as the gene of interest (GOI).

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

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
    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
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 733