Part:BBa_K1123005:Experience

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Applications of BBa_K1123005

As part of the iGEM 2013 competition the TU-Eindhoven team attempted to create a promoter which would induce protein expression under anaerobic conditions. This functional promoter could then be used to express CEST proteins (which should enable MRI imaging) upon the bacterium entering tumour regions. To test the functionality of the promoter we decided to express EGFP under anaerobic conditions. EGFP can be easily analysed making it easy for us to characterize the workings of the FNR promoter.

If we manage to express EGFP using this FNR promoter: BBa_K1123000 then we will be able to express other CEST proteins anaerobically with a high probability of success. BBa_K1123001, BBa_K1123002, BBa_K1123003, BBa_K1123004, BBa_K1123005, BBa_K1123007, BBa_K1123008, BBa_K1123009

For the EGFP expression under influence of the FNR promoter we cloned this biobrick into a pBR322 vector. This vector was then transformed into BL21 bacteria. Colonies of this transformation product were grown in (6) 8mL culture tubes and were then injected into a 4L LB solution, found in a bioreactor. After the optical density had reached 0.600 the chamber was constantly flooded with Nitrogen for over 24 hours, having samples taken every few hours during the day. After this anaerobic expression was complete the samples were analysed but no EGFP could be observed. A disappointing result. Upon closer investigation of the BL21 E.coli strain, we found that the FNR production, necessary for the proper functioning of the FNR promoter was absent.

In response to this lack of FNR within the BL21 bacteria it was decided that we attempt expression within a second E.coli strain. For this the XL-1-Blue strain was chosen. Before performing the actual experiments of course some research was performed to ensure that our assumptions were also true.

Once certain this strain was FNR compatible we transformed this biobrick, once again in the pBR322 vector, into XL-1-Blue E.coli bacteria. These were grown on agar and then picked, placed in 8mL of LB and then brought over into 4L of LB. Here they were allowed to grow aerobically until the Optical Density (O.D.) was 0.600 or higher.

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