Part:BBa_K1111005:Experience

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Applications of BBa_K1111005

We constructed this plasmid in order to use it as a positive control for promoters that are inducible and inserten in front of the same reporter gene (superfolded GFP from iGEM). As it is constitutively expressed, the fluorescence can be used at least as a qualitative comparison to promoters that are induced.

Gibson Assembly and Primers

We used Gibson Assembly to insert this constitutive promoter into the pSB1C3 Backbone. We used the biobrick BBa_I746908 where we remove the Arac promoter but left the superfolded GFP. This way, the latter was under control of our Cad promoter.

Figure 1: History of the Gibson Assembly including Primers

Sequencing

We sequenced both the Promoter and the superfolded GFP. Both sequencing results showed that there were no mutations in the sequences.

Figure 2: Sequencing result of the constitutive promoter using one foreward Primer that binds directly to the beginning of the promoter

Figure 3: Sequencing result of the superfolded GFP using one foreward Primer that binds to the VF2 bindning site in the pSB1C3 plasmid

Plating Transformants

We plated the transformants on agar plates containing the antibiotic Chloramphenicol. As one can see, the expression of superfolded GFP can be seen very nicely by the naked eye.

Figure 4: Two plates were used to grow the bacteria over night at 37°C. The next day they had turned bright green.

OD Measurements

We transformed cell with our plasmid and then measured their OD in different media with different pHs. We compared this data with the Fluorescence measurements and were able to conclude that the reason why GFP expression decreases at low pH is that the cells are actually dying.

Figure 5: OD measurements of transformed DH5 alpha cells in media at varying pH values.

Measuring GFP expression using a PlateReader

We plated bacteria with our plasmid onto a plate reader and then measured their fluorescence. The fluorescence was high in the beginning and then decreased. In the media with acidic pH the GFP expression stayed stable (pH 5.5) or decreased even further after a while. In the media with pH 7 and pH 8.5 the fluorescence first decreased a little bit an then increased again towards the end of the measurement. In all four cases, the fluorescence was high, even after 18h, it was even visible by the naked eye on the plate reader.

Figure 6: Fluorescence of transformed DH5-alpha cells at pH 5.5.

Figure 7: Fluorescence of transformed DH5-alpha cells at pH 6.5.

Figure 8: Fluorescence of transformed DH5-alpha cells at pH 7.

Figure 9: Fluorescence of transformed DH5-alpha cells at pH 8.5.

Figure 10: Plate used for the PlateReader experiment. The cell marked in a black rectangle are the ones containing the constitutive promoter.

Observing Fluorescence under the microscope

We let the cells grow in media with different pH values and then checked if the pH has an impact in the expression of GFP. We used these cells as positive controls for two of our other constructs, in order to be able to campare the fluorescence qualitatively.

Figure 11: Fluorescence of transformed DH5-alpha cells in a medium where we added a 10X HEPES buffer and LB in a 1:1 ratio

Figure 12: Fluorescence of transformed DH5-alpha cells in a medium where we added a water and LB in a 1:1 ratio

Figure 13:Fluorescence of transformed DH5-alpha cells in a medium where we added a 10X HEPES buffer and LB in a 1:1 ratio

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