Difference between revisions of "Part:BBa K1529321"

Revision as of 12:34, 19 October 2014

Prhl(RR)_GFP

This part is Prhl(RR) promoter regulating GFP. Prhl(RR) is a promoter that is activated by C4HSL-RhlR complex.
We improved Prhl (BBa_R0071) by changing a LuxR binding site of Plux (BBa_R0062) to a RhlR binding site. (Fig. 1.)
The bottom structures of LuxR and RhlR are similar to each other, so RhlR can also bind to Lux Box. (Fig. 2.)

We constructed this part to characterize the function of the Prhl(RR) promoter (BBa_K1529310), by inserting Prhl(RR) promoter upstream of a GFP coding sequence.

Fig. 1. Our newly designed promoter

Fig. 2. Difference between LuxR and RhlR

By using reporter cells that contain Prhl(RR)-GFP, we measured the fluorescence intensity of the cells induced by C4HSL.
Through an induction assay, we found that our new Prhl(RL) promoter was actually activated by C4HSL-RhlR complex and had high induced/not-induced ratio(Fig. 3.).
This means Prhl(RL) promoter has the higher expression level while keeping the low leakage.

We can say that Prhl(RL) promoter is the best improved Prhl promoter due to the advantages of less leak and higher expression level.

Fig. 3. Fluorescence intensity detected by flow cytometer

For more information, see [http://2014.igem.org/Team:Tokyo_Tech/Experiment/Prhl_reporter_assay our work in Tokyo_Tech 2014 wiki].

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 90
Illegal BamHI site found at 78
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 777

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1529321 short</partinfo>
-Prhl(RR) is a promoter that is activated by C4HSL.<br>
+This part is Prhl(RR) promoter regulating GFP.
-We improved Prhl (<partinfo>BBa_R0071</partinfo>)  by changing LuxR binding site of Plux (<partinfo>BBa_R0062</partinfo>)  to RhlR binding site. (Fig.1)<br>
+Prhl(RR) is a promoter that is activated by C4HSL-RhlR complex.  <br>
-The bottom structures of LuxR and RhlR are similar to each other, so RhlR can bind to Lux Box. (Fig.2)  <br>
+We improved Prhl (<partinfo>BBa_R0071</partinfo>)  by changing a LuxR binding site of Plux (<partinfo>BBa_R0062</partinfo>)  to a RhlR binding site. (Fig. 1.)<br>
+The bottom structures of LuxR and RhlR are similar to each other, so RhlR can also bind to Lux Box. (Fig. 2.)  <br>
-On the downstream of the promoter, <i>GFP</i> is inserted as a reporter.<br>
+We constructed this part to characterize the function of the Prhl(RR) promoter (<partinfo>BBa_K1529310</partinfo>), by inserting Prhl(RR) promoter upstream of a GFP coding sequence. <br>
 [[Image:titech2014_parts_rhl_promoter_main_Fig1.png|thumb|center|550px|<b>Fig. 1.</b> Our newly designed  promoter]]
 [[Image:titech2014_parts_luxR_and_rhlR_difference_main_Fig2.png|thumb|center|550px|<b>Fig. 2.</b> Difference between LuxR and RhlR]]
-To characterize the function of the <i>rhl(RR)</i> promoter (<partinfo>BBa_K1529320</partinfo>), we constructed this part Prhl(RR)_GFP (<partinfo>BBa_K1529321</partinfo>) by inserting ''rhl(RR)'' promoter upstream of a GFP coding sequence. <br>
-By using reporter cells that contain Prhl(RR)-GFP, we measured the fluorescence intensity of the cells induced by C4HSL (Fig. 2). <br>
-We saw that our new ''rhl(RR)'' promoter was actually activated by C4HSL through an induction assay (Fig. 3).<br>
-[[Image:titech2014_parts_GFP_assay_main_Fig.3.jpg|thumb|center|550px|<b>Fig. 3.</b> Fluorescence intensity detected by flow cytometer]]
+By using reporter cells that contain Prhl(RR)-GFP, we measured the fluorescence intensity of the cells induced by C4HSL. <br>
+Through an induction assay, we found that our new Prhl(RL) promoter was actually activated by C4HSL-RhlR complex and had high induced/not-induced ratio(Fig. 3.). <br>
+This means Prhl(RL) promoter has the higher expression level while keeping the low leakage. <br>
+We can say that Prhl(RL) promoter is the best improved Prhl promoter due to the advantages of less leak and higher expression level. <br>
+[[Image:Improved_Prhl(RL)_Promoter_Assay_Result.png|thumb|center|600px|<b>Fig. 3.</b> Fluorescence intensity detected by flow cytometer]]
 <br>