Difference between revisions of "Part:BBa K1970000:Experience"

 
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===Applications of BBa_K1970004===
 
===Applications of BBa_K1970004===
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===User Reviews===
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team:<b>NTNU 2016</b>
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===Applications of BBa_K608351===
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We tested this part together with an mCherry reporter ([https://parts.igem.org/Part:BBa_E1010]) controlled by a self-inhibiting RBS. The retrons in this part are set to produce msDNA that targets the inhibitory stem loop of this RBS thereby inducing translational activity from it (an thus production of mCherry). Additionally the
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E. Coli DH5α cells were transformed with the constructs above. Transformants were inoculated overnight in LB medium with kanamycin, at 37 °C and 255rpm. The 5 cultures were placed in a water bath at 37 °C. The water bath was gradually heated to 42 °C to avoid heat shocking the cells. Time courses of the fluorescence (abs: 580nm, em: 615nm) and OD of the cells were recorded (see the figure below).
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[[Image:24_10_1_fused.png|900px]]
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[[Image:24_10_2_fused.png|900px]]
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The four combinations of RBS and terminator around an mCherry reporter coupled to a CI repressor (this part). In a) and b) the RBS was clearly not effective, with the fluorescence being close to that of cells lacking an mCherry. The heat treatment did not have any obvious influence on the fluorescence of the cells. The RBS in c) and d) had relatively high fluorescence values, which changed by an order of magnitude with the terminator chosen. However, the heat treatment did not have any obvious positive influence in these cases either. In d) the influence was negative in fact.
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Therefore we concluded that this part is not a good choice for temperature transitions between 37 and 42 °C. This agrees with the experience gained on this part by other groups.
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===User Reviews===
 
===User Reviews===

Revision as of 12:24, 26 October 2016


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_K1970004

User Reviews

team:NTNU 2016

Applications of BBa_K608351

We tested this part together with an mCherry reporter ([1]) controlled by a self-inhibiting RBS. The retrons in this part are set to produce msDNA that targets the inhibitory stem loop of this RBS thereby inducing translational activity from it (an thus production of mCherry). Additionally the



E. Coli DH5α cells were transformed with the constructs above. Transformants were inoculated overnight in LB medium with kanamycin, at 37 °C and 255rpm. The 5 cultures were placed in a water bath at 37 °C. The water bath was gradually heated to 42 °C to avoid heat shocking the cells. Time courses of the fluorescence (abs: 580nm, em: 615nm) and OD of the cells were recorded (see the figure below).


24 10 1 fused.png

24 10 2 fused.png

The four combinations of RBS and terminator around an mCherry reporter coupled to a CI repressor (this part). In a) and b) the RBS was clearly not effective, with the fluorescence being close to that of cells lacking an mCherry. The heat treatment did not have any obvious influence on the fluorescence of the cells. The RBS in c) and d) had relatively high fluorescence values, which changed by an order of magnitude with the terminator chosen. However, the heat treatment did not have any obvious positive influence in these cases either. In d) the influence was negative in fact.


Therefore we concluded that this part is not a good choice for temperature transitions between 37 and 42 °C. This agrees with the experience gained on this part by other groups.


User Reviews

UNIQc58f13c5f4289430-partinfo-00000000-QINU UNIQc58f13c5f4289430-partinfo-00000001-QINU