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.
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Figure 1: pCold cells on LB+Amp plate after exposure to cold shock
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[[File:Bba M50470 plate image.jpeg|200px|center]]
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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.
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Figure 2 and 3: Dynamic Range Experiment for pCold
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[[File:Bba M50470 time course image one.jpeg|200px|center]]
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[[File:Bba M50470 time course image two.jpeg|200px|center]]
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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.
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Figure 4: Western Blot against 3X-FLAG tag
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[[File: Bba M50470 western blot image.jpeg|200px|center]]  
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Given the results of our dynamic range experiment, we performed a Western Blot analysis to confirm whether or not aeBlue was being produced by the bacteria. After cold shock at 4C for approximately 24 hours, we performed a Western blot. We used an anti-FLAG primary antibody which would bind to the 3X-FLAG tag present on this plasmid. As shown in Figure 4, we were able to confirm that aeBlue was being produced, however not in quantitative amounts that were visible to the naked eye.  Further design of this construct could include ways to increase the amount of protein expressed when exposed to cold, as well as eliminating the activity at 37C.

Latest revision as of 14:36, 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

UNIQf2292e060b82f915-partinfo-00000000-QINU UNIQf2292e060b82f915-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

Bba M50470 plate image.jpeg

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

Bba M50470 time course image one.jpeg
Bba M50470 time course image two.jpeg

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.

Figure 4: Western Blot against 3X-FLAG tag

Bba M50470 western blot image.jpeg

Given the results of our dynamic range experiment, we performed a Western Blot analysis to confirm whether or not aeBlue was being produced by the bacteria. After cold shock at 4C for approximately 24 hours, we performed a Western blot. We used an anti-FLAG primary antibody which would bind to the 3X-FLAG tag present on this plasmid. As shown in Figure 4, we were able to confirm that aeBlue was being produced, however not in quantitative amounts that were visible to the naked eye. Further design of this construct could include ways to increase the amount of protein expressed when exposed to cold, as well as eliminating the activity at 37C.