Difference between revisions of "Part:BBa K1602044"

Latest revision as of 21:26, 19 November 2015

RRL3H

Composite part consisting of a constitutive promoter (BBa_J23100), Lock3 (BBa_J01080) and hokD (BBa_K1497008). It belongs to a two-part killswitch-system for E.coli utillizing a riboregulator for posttransciptional regulation of hokD. Upon transcription the sequence of Lock3 forms a hairpin-secondary-structure masking the ribosome binding site (RBS) and therefore prevents translation of the following hokD-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 hokD resulting in cell death.

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

.

Functional Parameters

In order to assemble the final riboregulator-systems the parts RRL3H (BBa_K1602044) and RRK3 (BBa_K1602046) on two seperate plasmids (pSB1C3 and pSB1A2) were co-transformed into E.coli(Top10) for the first riboregulator. For the second riboregulator containing hokD the part RRlocked (BBa_K1602050) was co-transformed with araC-pBAD-RRkey (BBa_K1602051). Positive transformants were selected by using two antibiotics, Chloramphenicol and Ampicillin, and verified via colony-PCR. A strain of E.coli (TOP10) transformed with araCpBAD-hokD (BBa_K1602043) was used as positive control and as negative control for each riboregulator Top10 transformed with only the cis-repressed part (RRL3H and RRlocked) of each system. All five 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 an aliquot of each culture was diluted several times and different dillutions were spread on LB-agar plates containing 20mM glucose. The plates were incubated at 37°C over night until single colonies were distinquishable. The amount of colonies was counted in order to determine the colony forming units per ml culture (CFU/ml) to compare the amount of living bacteria in the cultures grown with glucose and arabinose.

**Figure 2:** Results of the spread plate assay used to determine the amount of CFU/ml for the cultures grown with either glucose (red) or arabinose (black)

The positive control (araC-pBAD-hokD) showed a significant reduction of CFU/ml in the culture grown with arabinose compared to the culture grown with glucose indicating that the presence of glucose repressed hokD-expression while the addition of arabinose leads to production of HokD resulting in increased cell death (Fig.2). The results for the riboregulators however did not turn out as expected. There was no significant reduction in the CFU/ml of the culture grown with arabinose compared to the culture grown with glucose observable for both riboregulator-systems (RRK3+RRL3H and araC-pBAD-RRkey+ RRlocked). This might indicate that the interaction between the taRNA and the crRNA does not occure as predicted resulting in a constant repression of hokD even when induced with arabinose.

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.

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
COMPATIBLE WITH RFC[1000]

@@ Line 16: / Line 16: @@
 ===Functional Parameters===
-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.
+In order to assemble the final riboregulator-systems the parts RRL3H (<html><a href="/Part:BBa_K1602044">BBa_K1602044</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) for the first riboregulator. For the second riboregulator containing hokD the part RRlocked (<html><a href="/Part:BBa_K1602050">BBa_K1602050</a></html>) was co-transformed with araC-pBAD-RRkey (<html><a href="/Part:BBa_K1602051">BBa_K1602051</a></html>). Positive transformants were selected by using two antibiotics, Chloramphenicol and Ampicillin, and verified via colony-PCR.
+A strain of <i>E.coli</i> (TOP10) transformed with araCpBAD-hokD (<html><a href="/Part:BBa_K1602043">BBa_K1602043</a></html>) was used as positive control and as negative control for each riboregulator Top10 transformed with only the cis-repressed part (<html><a href="/Part:BBa_K1602044">RRL3H</a></html> and <html><a href="/Part:BBa_K1602050">RRlocked</a></html>) of each system.
-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.
+All five 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 an aliquot of each culture was diluted several times and different dillutions were spread on LB-agar plates containing 20mM glucose. The plates were incubated at 37°C over night until single colonies were distinquishable. The amount of colonies was counted in order to determine the colony forming units per ml culture (CFU/ml) to compare the amount of living bacteria in the cultures grown with glucose and arabinose.
 <html>
 <center>
 <figure >
-	<img width=50%; src="https://static.igem.org/mediawiki/parts/8/86/FACS-registry-RRL3G_final.png">
+	<img width=50%; src="https://static.igem.org/mediawiki/parts/8/8b/Spread1.PNG">
-	<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>
+	<figcaption><b>Figure 2:</b> Results of the spread plate assay used to determine the amount of CFU/ml for the cultures grown with either glucose (red) or arabinose (black)</figcaption>
 </figure>
 </center>
 </html>
-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.
+The positive control (araC-pBAD-hokD) showed a significant reduction of CFU/ml in the culture grown with arabinose compared to the culture grown with glucose indicating that the presence of glucose repressed hokD-expression while the addition of arabinose leads to production of HokD resulting in increased cell death (Fig.2). The results for the riboregulators however did not turn out as expected. There was no significant reduction in the CFU/ml of the culture grown with arabinose compared to the culture grown with glucose observable for both riboregulator-systems (RRK3+RRL3H and araC-pBAD-RRkey+ RRlocked). This might indicate that the interaction between the taRNA and the crRNA does not occure as predicted resulting in a constant repression of hokD even when induced with arabinose.
-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 (<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.
 ===References===
+<!-- -->