Difference between revisions of "Part:BBa K634007:Experience"
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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: | ||
− | Reporter | + | '''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). | |
− | Counter selection | + | '''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=== | ===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
UNIQ45683ad3629669da-partinfo-00000000-QINU UNIQ45683ad3629669da-partinfo-00000001-QINU