Difference between revisions of "Part:BBa K634007:Experience"

(→‎Applications of BBa_K634007)
(→‎Applications of BBa_K634007)
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When placed 3' of the promoter portion of a 1- or 2-component sensor, BBa_634007 can be used in the following ways:  
 
When placed 3' of the promoter portion of a 1- or 2-component sensor, BBa_634007 can be used in the following ways:  
  
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Reporter: tagRFP (BBa_K634005) fluoresces red upon induction (see BBa_K634005 for issues with fluorescence)
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'''Reporter'''
  
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Selection: Sensor systems can be selected for desired induced expression through supplementation of the culture with the sensor's analyte and kanamycin.  
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tagRFP ([[BBa_K634005]]) fluoresces red upon induction. This can be useful for visually verifying expression of the cassette is proceeding normally, first order approximations of promoter activity, and (with plate reader analysis) as a marker for quantification of expression. Cell cultures have not appeared visibly red in our experience, but are easy to see when pelleted (see example).  
  
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Counter selection: Sensors can also be selected for absence of expression when uninduced through supplementation of the culture with sucrose
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'''Selection'''
 +
 
 +
Sensor systems can be selected for desired induced expression through supplementation of the culture with the sensor's analyte and kanamycin.
 +
 
 +
'''Counter selection'''
 +
 
 +
Sensors can also be selected for absence of expression when uninduced through supplementation of the culture with sucrose
 +
 
 +
 
 +
== Issues in practice ==
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''sacB'' appears to be something of a lightning rod for loss of function mutations. As can be seen below, sucrose lethality is only maintained in a population for approximately 5 hours. At this point, we presume that spontaneous mutations in ''sacB'' produce sucrose-insensitive mutants.
  
 
===User Reviews===
 
===User Reviews===

Revision as of 03:59, 27 September 2011

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.

Applications of BBa_K634007

When placed 3' of the promoter portion of a 1- or 2-component sensor, BBa_634007 can be used in the following ways:

Reporter

tagRFP (BBa_K634005) fluoresces red upon induction. This can be useful for visually verifying expression of the cassette is proceeding normally, first order approximations of promoter activity, and (with plate reader analysis) as a marker for quantification of expression. Cell cultures have not appeared visibly red in our experience, but are easy to see when pelleted (see example).

Selection

Sensor systems can be selected for desired induced expression through supplementation of the culture with the sensor's analyte and kanamycin.

Counter selection

Sensors can also be selected for absence of expression when uninduced through supplementation of the culture with sucrose


Issues in practice

sacB appears to be something of a lightning rod for loss of function mutations. As can be seen below, sucrose lethality is only maintained in a population for approximately 5 hours. At this point, we presume that spontaneous mutations in sacB produce sucrose-insensitive mutants.

User Reviews

UNIQed2042c6b42c7654-partinfo-00000000-QINU UNIQed2042c6b42c7654-partinfo-00000001-QINU