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Part:BBa_K1111004:Experience

Designed by: Luisa Spisak   Group: iGEM13_EPF_Lausanne   (2013-09-19)
Revision as of 19:04, 2 October 2013 by GigiGoesGugu (Talk | contribs) (→‎Measuring Fluorescence with a PlateReader)

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Applications of BBa_K1111004

We wanted to test if the promoter that we cloned into the plasmid really can be induced upon external acidification. In order to do that, we transformed bacteria with it, incubated them at different pH and then measured their GFP expression using a plate reader.

Gibson Assembly and Primers

We used Gibson Assembly to insert the Cad 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

The sequencing result showed that there were no mutations in the insert

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

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 is not expressed at low pH is that the bacteria are actually dying in these two media


Figure 3: Growth Curve of transformed DH5-alpha cells at different pH values.

Measuring Fluorescence with a PlateReader

We wanted to know if the promoters were indeed induced at low pH. For this we let the bacteria grow in differenly buffered media in aplate reader and the measured their Fluorescence.


Figure 4: Fluorescence of transformed DH5-alpha cells at pH 5.5.
Figure 5: Fluorescence of transformed DH5-alpha cells at pH 6.5.
Figure 6: Fluorescence of transformed DH5-alpha cells at pH 7.


Figure 7: Fluorescence of transformed DH5-alpha cells at pH 8.5.

Observing Fluorescence under the Microscope

We wanted to induce GFP expression in our bacteria by adding different buffers to the media. We then let them grow for a while and then looked at them under the microscope to see if they were expressing GFP or not. We compared their fluorescence with another biobrick we made in which we put a constitutive promoter in fron of superfolded GFP as a qualitative measure.

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


Figure 1: Fluorescence of transformed DH5-alpha containing a constitutive promoter in a media with a 1:1 ratio of LB and water

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