Part:BBa_M50477:Experience

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. 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 indefinitely at varying heat temperatures . The tubes were tested for apparent meffRed production after 30 mintues and 60 minutes. As shown in Figure 1, we failed to see any meffRed after 60 minutes at any temperature; this caused us to terminate the experiment and conclude that insufficient meffRed was being produced to visually verify its production

Figure 1: Dynamic range experiment for pHeat

In order to rule out incorrect an incorrect pHeat sequence as the culprit of our results, we performed PCR and subsequently gel electrophoresis on pHeat-transformed E. coli cells. As shown in Figure 2, PCR of three different colonies of pHeat-transformed bacteria all yielded a DNA sequence of the same length, suggesting that the DNA sequence was, in fact, correct.

Figure 2: pHeat gel electrophoresis

Given the results of our dynamic range experiment, we performed a Western Blot analysis to confirm that meffRed was, in fact, not being produced by the bacteria. After 5 minutes of heat shock in 42C, we performed a Western blot. We used an anti-His primary antibody which would bind to the His tag present on this plasmid. As shown in Figure 3, we confirmed that no meffRed was produced after 5 minutes of heat-shock. Further experiments could include performing a reverse transcription assay to determine if any mRNA is being produced after heat-shock.

Figure 3: Western blot analysis of meffRed production after brief heat-shock period (at 42C).