Part:BBa_J100205:Experience

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Applications of BBa_J100205

Best Growth Conditions: J100205 (repClone Red) was used to build part BBa_J100306, repClone Red with wt tetR promoter. We transformed E. coli with plasmids containing J100306 or J100308 (a variant of J100306) and measured the induction caused by the addition of 200 ng/mL anhydrous tetracycline (aTc) to determine the incubation time needed for maximum induction. We found that aTc is most effective after fourteen to sixteen hours of growth, after which the induction is lost.

We performed an aTc dose-response test on E. coli containing part J100306 to determine the ideal concentration of aTc for induction of RFP. We found that 400 ng/mL aTc gives the best compromise of fold induction and allowing for sufficient cell growth. Induction above 400 ng/mL looks higher because fewer cells grew and therefore the denominator was smaller when calculating the ratio.

Results of Dose Response Assay:

Results of Time Assay:

Positive and Negative Controls for TetR Promoter Editing: We used J100205 to build part BBa_J100312, repClone Red with scrambled TetR promoter. We transformed E. coli with plasmids containing J100312 or J100306 (another J100205 variant, with wt promoter) and measured induction caused by addition of 400 ng/mL aTc. We found that a scrambled promoter allows non-repressible transcription of RFP. As shown above, the wildtype promoter was repressible, and the presence of aTc resulted in a fold change greater than 100x. When using J100205 to test variants of the TetR promoter, J100306 can be used as a positive control (repressible) and J100312 as a negative control (non-repressible).

Fluorescence by J100306 without aTc does not appear on the graph but was quantifiable. See data table below.

The numerical data are displayed, including the raw data for each treatment and ratio of RFU with aTc to RFU without aTc (a value of 1 would mean no change).

The ratios were calculated by dividing RFU with aTc by RFU without aTc for each construct.

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