Difference between revisions of "Part:BBa M50470:Experience"
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| + | We transformed competent E. coli cells with this heat-shock plasmid. After growing the transformed cells on LB + ampicillin agar plates, we grew individual colonies in 14mL round bottom tubes with LB + ampicillin. | ||
| + | |||
| + | Figure 1: pCold cells on LB+Amp plate after exposure to cold shock | ||
[[File:Bba M50470 plate image.jpeg|200px|center]] | [[File:Bba M50470 plate image.jpeg|200px|center]] | ||
| − | + | For our first assay, we exposed pCold to cold shock conditions by placing plates in a 4C cold room for an extended period of time. Our first plate yielded only a few blue colonies after two or so weeks, so while promising, this data was not conclusive due to the time frame, as well as contamination of another erroneous plasmid during transformation and/or plating. We then grew another plate and while we did obtain blue by following the exact protocol as before, the cells were blue once they were removed from the 37C incubator, indicating that this promoter is active at both 37C and 15C. | |
| + | Figure 2 and 3: Dynamic Range Experiment for pCold | ||
[[File:Bba M50470 time course image one.jpeg|200px|center]] | [[File:Bba M50470 time course image one.jpeg|200px|center]] | ||
[[File:Bba M50470 time course image two.jpeg|200px|center]] | [[File:Bba M50470 time course image two.jpeg|200px|center]] | ||
| − | + | For our dynamic range experiment, we placed 1.5 mL of transformed cells and tested them in triplicates under the following thermocycler conditions: 5 minutes at 37C and then indefinitely at varying cold temperatures . The tubes were tested for apparent aeBlue production after 30 mintues, 60 minutes, and 90 minutes. We failed to see any aeBlue after 90 minutes at any temperature which caused us to terminate the experiment and speculate that insufficient aeBlue was being produced to visually verify its production. | |
| + | |||
[[File: Bba M50470 western blot image.jpeg|200px|center]] | [[File: Bba M50470 western blot image.jpeg|200px|center]] | ||
| − | + | To determine | |
Revision as of 14:31, 12 December 2018
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_M50470
This part is intended to be part of a "visual thermometer" temperature sensor for which the color expressed by the color expressed by the Escherichia coli reflect the temperature of the surrounding environment. This part specifically responds to cold temperatures, however, we've found that it responds variably and slowly, and is also active at 37C, which could not be classified as a cold temperature.
Stanford Location
Stanford Location Plasmid name: pCold Organism: E. coli Device type: Sensor Glycerol stock barcode: 0133024503 Box label: BioE44 F18 Location: -80C Freezer, A4
User Reviews
UNIQ8d378d61d19b54be-partinfo-00000000-QINU UNIQ8d378d61d19b54be-partinfo-00000001-QINU
We transformed competent E. coli cells with this heat-shock plasmid. After growing the transformed cells on LB + ampicillin agar plates, we grew individual colonies in 14mL round bottom tubes with LB + ampicillin.
Figure 1: pCold cells on LB+Amp plate after exposure to cold shock
For our first assay, we exposed pCold to cold shock conditions by placing plates in a 4C cold room for an extended period of time. Our first plate yielded only a few blue colonies after two or so weeks, so while promising, this data was not conclusive due to the time frame, as well as contamination of another erroneous plasmid during transformation and/or plating. We then grew another plate and while we did obtain blue by following the exact protocol as before, the cells were blue once they were removed from the 37C incubator, indicating that this promoter is active at both 37C and 15C.
Figure 2 and 3: Dynamic Range Experiment for pCold
For our dynamic range experiment, we placed 1.5 mL of transformed cells and tested them in triplicates under the following thermocycler conditions: 5 minutes at 37C and then indefinitely at varying cold temperatures . The tubes were tested for apparent aeBlue production after 30 mintues, 60 minutes, and 90 minutes. We failed to see any aeBlue after 90 minutes at any temperature which caused us to terminate the experiment and speculate that insufficient aeBlue was being produced to visually verify its production.
To determine