Team:SDU-Denmark/project-p

From 2010.igem.org

(Difference between revisions)
(Characterization)
(Characterization)
Line 251: Line 251:
[[Image:Team-SDU-Bielefeld.PNG|400px|thumb|OD550nm of MG1655-pSB1C3-K389016 taken every 2 hours for a 10 hour period. All data can be seen under [https://static.igem.org/mediawiki/2010/5/5b/Team-SDU-Bielefeld_karakterisering.ZIP Raw data]]]<br><br>
[[Image:Team-SDU-Bielefeld.PNG|400px|thumb|OD550nm of MG1655-pSB1C3-K389016 taken every 2 hours for a 10 hour period. All data can be seen under [https://static.igem.org/mediawiki/2010/5/5b/Team-SDU-Bielefeld_karakterisering.ZIP Raw data]]]<br><br>
The Bielefeld team wanted us to verify their characterization results of K389016 (VirA/G reporter device mRFP). The characterisation results were carried out according to protocol ([https://2010.igem.org/Team:SDU-Denmark/protocols#Charactarization_of_K389016_.28VirA.2FG_reporter_device_mRFP.29 CK1.1]). Different cultures were made with acetosyringone concentrations of 100uM, 200uM and 400uM. A culture with 0uM acetosyringone was used as a control. The cultures were incubated at 37 degrees Celsius, until the cells reached stationary phase. A growth assay was carried out with measurements taken at OD<sub>550</sub>, every 2 hours for 10 hours. Parallel with the OD<sub>550</sub> measurements samples were taken and used for fluorescence measurements.<br><br>
The Bielefeld team wanted us to verify their characterization results of K389016 (VirA/G reporter device mRFP). The characterisation results were carried out according to protocol ([https://2010.igem.org/Team:SDU-Denmark/protocols#Charactarization_of_K389016_.28VirA.2FG_reporter_device_mRFP.29 CK1.1]). Different cultures were made with acetosyringone concentrations of 100uM, 200uM and 400uM. A culture with 0uM acetosyringone was used as a control. The cultures were incubated at 37 degrees Celsius, until the cells reached stationary phase. A growth assay was carried out with measurements taken at OD<sub>550</sub>, every 2 hours for 10 hours. Parallel with the OD<sub>550</sub> measurements samples were taken and used for fluorescence measurements.<br><br>
-
 
-
The fluorescence measurements was carried out on a Perkin Elmer LS55 fluorescence spectrometer. We were unable to detect any RFP in the cells. We furthermore tested the cells on a Typhoon Trio but once again we could not detect RFP. We duplicated the experiment three times, but none were successful. <br>
 
[[Image:Team SDU-Denmark Bielefeld Typhoon trio.JPG|210px|thumb|left|Typhoon trio fluorescence scanning of MG1655-pSB1C3-K389016 induced with 200 uM acetosyringone. Excitation: 633nm Emittance: 670nm. No RFP detection, any red seen in the picture is noice.]]<br>
[[Image:Team SDU-Denmark Bielefeld Typhoon trio.JPG|210px|thumb|left|Typhoon trio fluorescence scanning of MG1655-pSB1C3-K389016 induced with 200 uM acetosyringone. Excitation: 633nm Emittance: 670nm. No RFP detection, any red seen in the picture is noice.]]<br>
 +
The fluorescence measurements was carried out on a Perkin Elmer LS55 fluorescence spectrometer. We were unable to detect any RFP in the cells. We furthermore tested the cells on a Typhoon Trio but once again we could not detect RFP. We duplicated the experiment three times, but none were successful. <br><br>
We were advised by the Bielefeld team to use excitation 584nm and emittance 607nm. This was done when using the Perkin Elmer LS55 fluorescence spectrometer, but the Typhoon trio has limited settings and according to the Typhoon trio manual RFP can be detected at excitation 633nm and emittance 670nm. <br>  
We were advised by the Bielefeld team to use excitation 584nm and emittance 607nm. This was done when using the Perkin Elmer LS55 fluorescence spectrometer, but the Typhoon trio has limited settings and according to the Typhoon trio manual RFP can be detected at excitation 633nm and emittance 670nm. <br>  
The picture above shows the fluorescence scanning prefomed on the Bielefeld part induced with 200uM acetosyringone. We spinned an over night culture of MG1655-pSB1C3-K38901 down, removed the supernatant and resuspended the pellet, the resupension was transfered to a glass slide and covered by a cover slide. No emittans is seen in the picture, any light seen on the picture is noice. <br><br>
The picture above shows the fluorescence scanning prefomed on the Bielefeld part induced with 200uM acetosyringone. We spinned an over night culture of MG1655-pSB1C3-K38901 down, removed the supernatant and resuspended the pellet, the resupension was transfered to a glass slide and covered by a cover slide. No emittans is seen in the picture, any light seen on the picture is noice. <br><br>

Revision as of 13:13, 27 October 2010