Difference between revisions of "Part:BBa K3463017:Experience"

Revision as of 16:02, 26 October 2020

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Characterization BBa_K3463017

TTo validate the UTR designer prediction and check the B0017 relevance, we measured the fluorescence and the absorbance of the cultures of the strains containing one of the following constructs: PtacHigh-eGFP, PtacLow-eGFP, PtacHigh-eGFP-B0017 BBa_K3463018, PtacLow-eGFP-B0017 BBa_K3463017 , and a non-transformed strain as a negative control for 4,5h. The experiment was carried out 3 times.

We obtained the following result shown in figure 1.

Figure 1 Effect of UTR prediction and B0017 terminator on eGFP fluorescence expression. The Figure 5 shows that no fluorescence was detected in the culture of the control non- transformed strain. As predicted by UTR designer the strong 5’UTR sequence (high) gave higher fluorescent levels than the weak 5’UTR sequence (Low). The terminator B0017 seems to have a limited effect on the fluorescent levels. The last seems to only impact the eGFP production of the strong UTR sequence.

User Reviews

UNIQ8d0249f2d16d7ce5-partinfo-00000000-QINU UNIQ8d0249f2d16d7ce5-partinfo-00000001-QINU

@@ Line 6: / Line 6: @@
 ===Characterization BBa_K3463017===
-To evaluate if our system works, we performed 6 bacterial cultures of transformed <i>E. coli</i> Nissle BBa_K3463019 incubated with increasing amounts of synthetic BHL in the medium from 10nM to 100µM. Once all the media have been set up at T0 with 100µM / 10µM / 1µM / 100nM / 10nM and without BHL, bacteria were inoculated at OD600nm=0.1.Then, fluorescence measurements of eGFP were performed for 15H (hourly in triplicata until 7H) in opaque 96-wells. For each curve in figure 1, the first measurement point (T0) was set at 0 and subtracted to the following ones.
+TTo validate the UTR designer prediction and check the B0017 relevance, we measured the fluorescence and the absorbance of  the cultures of the strains containing one of the
-This experiment was repeated 3 times.
+following constructs: PtacHigh-eGFP, PtacLow-eGFP, PtacHigh-eGFP-B0017 BBa_K3463018, PtacLow-eGFP-B0017 BBa_K3463017 , and a non-transformed strain as a negative control for 4,5h. The experiment was carried out 3 times.
-[[Image:T--Grenoble Alpes--Res Survival BHL fluo.png|600px|thumb|center| '''Figure 1''' eGFP expression according to the BHL concentration and over time. Different concentrations of BHL were performed to evaluate the effect of BHL in E. coli Nissle. Thanks to the BHL, the engineered E. coli should be able to express eGFP. Without BHL, no eGFP expression should be detected.]]
+We obtained the following result shown in figure 1.
-Averages : 100µM/10µM/1µM > others
+[[Image:T--Grenoble Alpes--Res Survival Ptac construction.png|600px|thumb|center| '''Figure 1''' Effect of UTR prediction and B0017 terminator on eGFP fluorescence expression.
+The Figure 5 shows that no fluorescence was detected in the culture of the control non-
-The Figure 1 shows the fluorescence intensity of each bacterial culture according to the BHL concentration and over time. While the wild type <i>E. coli</i> Nissle auto-fluorescence is used as blank, all the other transformed cultures show significant eGFP expression.
+transformed strain. As predicted by UTR designer the strong 5’UTR sequence (high) gave higher fluorescent levels than the weak 5’UTR sequence (Low). The terminator B0017 seems to have a limited effect on the fluorescent levels. The last seems to only impact the eGFP production of the strong UTR sequence.]]
-First, we can see that transformed <i>E. coli</i> Nissle produce low quantities of eGFP even without BHL. However, we can also observe that the more BHL, the more eGFP expression (p-value<2.10-16). Indeed, in the culture containing 10µM or 100µM of BHL, the fluorescent intensity starts to increase sharply after 3 hours of culture and reaches 600 or more. Nevertheless, even if  curves reach 400 and 100 with 1µM and 100nM respectively, weaker concentrations of BHL (10nM) don't allow transformed <i>E. coli</i> Nissle to significantly express eGFP.
 ===User Reviews===