Difference between revisions of "Part:BBa J09855:Experience"

Latest revision as of 10:39, 18 September 2015

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.

Further testing of BBa_J09855 of Technion HS 2015 team

AHLs, (N-acylhomoserine lactones), known as autoinducers (AIs), are widely conserved signal molecules present in quorum-sensing systems of many gram-negative bacteria. The conservation and potent activity of these signaling molecules is based on the lactone ring. LuxR,is a protein that can bind AHL. When bound to AHL it can stimulate transcription by binding to LuxR binding sites of the lux promoter (pLux). The well studied operon of the pLux promoter is originally isolated from the bacterium Vibrio fischeri. . The tet promoter is constitutively active and can be repressed by the transcriptional regulator TetR. In turn, TetR repression is inhibited by the addition of tetracycline. Here is a scheme that helps understand how the BioBrick works:

In 2015, the HS iGEM team generated and tested the part BBa_K1767010 that is based on this existing BioBrick. Compared to other teams, we measured fluorescent protein expression of 600 minutes.

Applications of BBa_J09855

This part is tested by 2012 iGEM team Tsinghua.

We use this part to work as a promoter system which can be activated by C6HSL. We can add some other gene dowmstream so that AHL can work as a signal. In our project this year, we use this part and modified it to get more complicated construct.

User Reviews

UNIQ2df5aa423dd824bb-partinfo-00000000-QINU UNIQ2df5aa423dd824bb-partinfo-00000001-QINU This part is tested by 2012 iGEM team Tsinghua.

To tell the truth, when we use this part for the first time, it didn't work well. Then we sequenced the part using prefix and suffix primer. We found that there are some part missed in Plac promoter at the beginning and in Plux promoter at the end. Maybe this is why it can't work.

We improve this part and modify sequence. We add several nucleic acid to the Plac promoter and plux promoter. This modification improve the efficiency of transcription. The sequensing results show that our construct is correct.

Also, we add RFP at the downstream of Plux promoter. This fluorescence protein is very easy to detect so that we can use this part to test AHL and work as reporter. Our new part is BBa_K766003.

This part was tested by ETH Zurich-iGEM 2013
The BBa_J09855 was used as a LuxR-generating construct together with GFP BBa_E0840 expressed from P_LuxR BBa_R0062 to build a sensor system for AHL. The construct was tested in LB liquid cultures over 16 h without and with induction through AHL (100 nM). The GFP fluorescence was measured using a TECAN plate reader and normalized to the OD₆₀₀.

The normalized GFP fluorescence of the AHL inducible GFP system based on BBa_J09855.

@@ Line 2: / Line 2: @@
 This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 how you used this part and how it worked out.
+===Further testing of BBa_J09855 of Technion HS 2015 team===
+AHLs, (N-acylhomoserine lactones), known as autoinducers (AIs), are widely conserved signal molecules present in quorum-sensing systems of many gram-negative bacteria. The conservation and potent activity of these signaling molecules is based on the lactone ring. LuxR,is a protein that can bind AHL. When bound to AHL it can stimulate transcription by binding to LuxR binding sites of the lux promoter (pLux). The well studied operon of the pLux promoter is originally isolated from the bacterium Vibrio fischeri. . The tet promoter is constitutively active and can be repressed by the transcriptional regulator TetR. In turn, TetR repression is inhibited by the addition of tetracycline.
+Here is a scheme that helps understand how the BioBrick works:
+[[File: Technion_HS_Israel_K1767010.jpg|500px|center]]
+In 2015, the HS iGEM team generated and tested the part BBa_K1767010 that is based on this existing BioBrick. Compared to other teams, we measured fluorescent protein expression of 600 minutes.
+[[File: Technion HS Israel K1767010 result.png|500px|center]]
 ===Applications of BBa_J09855===
-We use this part to work as a promoter system which can be activated by C6HSL. We can add some other gene dowmstream so that AHL can work as a signal. In our project this year(Team: Tsinghua), we use this part and modified it to get more complicated construct.
+This part is tested by 2012 iGEM team '''Tsinghua.'''
+We use this part to work as a promoter system which can be activated by C6HSL. We can add some other gene dowmstream so that AHL can work as a signal. In our project this year, we use this part and modified it to get more complicated construct.
 ===User Reviews===
@@ Line 19: / Line 31: @@
 <!-- End of the user review template -->
 <!-- DON'T DELETE --><partinfo>BBa_J09855 EndReviews</partinfo>
+This part is tested by 2012 iGEM team '''Tsinghua.'''
+To tell the truth, when we use this part for the first time, it didn't work well. Then we sequenced the part using prefix and suffix primer. We found that there are some part missed in Plac promoter at the beginning and in Plux promoter at the end. Maybe this is why it can't work.
+We improve this part and modify sequence. We add several nucleic acid to the Plac promoter and plux promoter. This modification improve the efficiency of transcription. The sequensing results show that our construct is correct.
+Also, we add RFP at the downstream of Plux promoter. This fluorescence protein is very easy to detect so that we can use this part to test AHL and work as reporter. Our new part is BBa_K766003.
+<br><br>This part was tested by '''ETH Zurich-iGEM 2013'''<br>
+The BBa_J09855 was used as a LuxR-generating construct together with GFP [https://parts.igem.org/Part:BBa_E0840 BBa_E0840] expressed from P<sub>LuxR</sub> [https://parts.igem.org/Part:BBa_R0062 BBa_R0062] to build a sensor system for AHL. The construct was tested in LB liquid cultures over 16 h without and with induction through AHL (100 nM). The GFP fluorescence was measured using a TECAN plate reader and normalized to the OD<sub>600</sub>.
+[[File:ETHZ J09855registryentry2.png|900px|center|thumb| <b>The normalized GFP fluorescence of the AHL inducible GFP system based on BBa_J09855.</b>]]