Difference between revisions of "Part:BBa K1073035:Experience"

Latest revision as of 08:46, 17 October 2013

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Applications of BBa_K1073035

The iGEM Team Braunschweig 2013 used this device to control growth of E. coli Top10 F' in ampicillin (0.29 mM) containing complex medium by inducing expression of ampR by either adding synthetic autoinducer N-butyryl homoserine lactone or cultivating the cells together with a strain containing device BBa_K1073034.

User Reviews

UNIQc27ff579de6e50d6-partinfo-00000001-QINU

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iGEM Team Braunschweig 2013

Using ampicillin containing complex medium, growth of cells containing this device was induced by adding synthetic autoinducer N-butyryl homoserine lactone to the culture broth. The required transcription regulator RhlR is encoded in the device. Cells also exhibit a bright pink color due to the constitutively expressed eforRed chromoprotein. Thus eforRed was used as a selection marker identifying cells containing this device. 2xYT medium containing ampicillin was used for cultivation and cells were incubated at 37°C and 250 rpm in non-baffled flasks.

iGEM Team Braunschweig 2013: Comparison of growth curves of E. coli Top10 F' containing BBa_K1073035 on the high copy plasmid pSB1C3 in the presence and absence of autoinducer N-butyryl-HSL in the culture broth.

Due to the leakiness of the las promoter the beta lactamase gene ampR is also expressed to some extend when no N-butyryl-HSL is added to the culture broth. However, expression is much higher when the autoinducer is added. We suggest that when there are no autoinducer molecules present in the culture broth growth of cells does not start until all ampicillin is degrated by the background activity of the beta lactamase.

We could not detect cross induction of the rhl promoter by RhlR and N-3-oxododecanoyl-HSL which is produced by LasI encoded in the device.

However, growth of cells could also be induced when culture supernatant of BBa_K1073034 containing N-butyryl-HSL synthesized by RhlI encoded in BBa_K1073034 is added to cultures on agar plates containing ampicillin.

iGEM Team Braunschweig 2013: Growth of E. coli Top10 containing BBa_K1073035 on the high copy plasmid pSB1C3 induced by the supernatant of expressed BBa_K1073034. The supernatant contains N-butyryl-HSL produced by RhlI. A piece of filter paper was soaked with supernatant of a BBa_K1073034 and placed on the agar plate containing ampicillin (0.29 mM).

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 ===Applications of BBa_K1073035===
-The iGEM Team Braunschweig 2013 used this device to control growth of ''E. coli Top10'' in ampicillin (0.29 mM) containing complex medium by inducing expression of ''ampR'' by either adding synthetic autoinducer N-buturyl homoserine lactone or cultivating the cells together with a strain containing device <partinfo>K1073034</partinfo>.
+The iGEM Team Braunschweig 2013 used this device to control growth of ''E. coli Top10 F''' in ampicillin (0.29 mM) containing complex medium by inducing expression of ''ampR'' by either adding synthetic autoinducer N-butyryl homoserine lactone or cultivating the cells together with a strain containing device <partinfo>K1073034</partinfo>.
 ===User Reviews===
@@ Line 16: / Line 16: @@
 <I>iGEM Team Braunschweig 2013</I>
 |width='60%' valign='top'|
-Using ampicillin containing complex medium, growth of cells containing this device could be induced by adding synthetic autoinducer N-buturyl homoserine lactone to the culture broth. The required transcription regulator RhlR is encoded in the device. Cells also exhibit a bright pink color due to the constitutively expressed eforRed chromoprotein. Thus eforRed was used as a selection marker identifying cells containing this device.
+Using ampicillin containing complex medium, growth of cells containing this device was induced by adding synthetic autoinducer N-butyryl homoserine lactone to the culture broth. The required transcription regulator RhlR is encoded in the device. Cells also exhibit a bright pink color due to the constitutively expressed eforRed chromoprotein. Thus eforRed was used as a selection marker identifying cells containing this device.
-xYT medium containing ampicillin was used for cultivation and cells were incubated at 37°C and 250 rpm.
+xYT medium containing ampicillin was used for cultivation and cells were incubated at 37°C and 250 rpm in non-baffled flasks.
-[[Image:Braunschweig_2013_-_Top10_pSB1C3_K1073035.jpg|420px]]
+[[Image:Braunschweig2013_Top10_pSB1C3_K1073035.jpg|500px]]
 '''iGEM Team Braunschweig 2013:'''
-Comparison of growth curves of ''E. coli Top10'' containing BBa_K1073035 on the high copy plasmid pSB1C3 in the presence and absence of autoinducer N-buturyl-HSL in the culture broth.
+Comparison of growth curves of ''E. coli Top10 F' '' containing BBa_K1073035 on the high copy plasmid pSB1C3 in the presence and absence of autoinducer N-butyryl-HSL in the culture broth.
-Due to the leakiness of the las promoter the beta lactamase gene ''ampR'' is also expressed to some extend when no N-buturyl-HSL is added to the culture broth. However, expression is much higher when the autoinducer is added.
+Due to the leakiness of the las promoter the beta lactamase gene ''ampR'' is also expressed to some extend when no N-butyryl-HSL is added to the culture broth. However, expression is much higher when the autoinducer is added. We suggest that when there are no autoinducer molecules present in the culture broth growth of cells does not start until all ampicillin is degrated by the background activity of the beta lactamase.
-Growth of cells could also be induced when culture supernatant of <partinfo>BBa_K1073034</partinfo> containing N-buturyl-HSL synthesized by RhlI encoded in BBa_K1073034 is added to cutures on agar plates containing ampicillin.
+We could not detect cross induction of the rhl promoter by RhlR and N-3-oxododecanoyl-HSL which is produced by LasI encoded in the device.
+However, growth of cells could also be induced when culture supernatant of <partinfo>BBa_K1073034</partinfo> containing N-butyryl-HSL synthesized by RhlI encoded in BBa_K1073034 is added to cultures on agar plates containing ampicillin.
 [[Image:Braunschweig_2013_-_K1073035_induced.png|250px]]
 '''iGEM Team Braunschweig 2013:'''
-Growth of ''E. coli Top10'' containing BBa_K1073035 on the high copy plasmid pSB1C3 induced by the supernatant of expressed BBa_K1073034. The supernatant contains N-buturyl-HSL produced by RhlI. A piece of filter paper was soaked with supernatant of a BBa_K1073034 and placed on the agar plate containing ampicillin.
+Growth of ''E. coli Top10'' containing BBa_K1073035 on the high copy plasmid pSB1C3 induced by the supernatant of expressed BBa_K1073034. The supernatant contains N-butyryl-HSL produced by RhlI. A piece of filter paper was soaked with supernatant of a <partinfo>BBa_K1073034</partinfo> and placed on the agar plate containing ampicillin (0.29 mM).
 |};
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