Part:BBa_K2669000:Experience

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iGEM Uppsala 2018 Experience

After transforming our cells with a low copy amplicilin plasmid containing this composite part, cell lysis and affinity chromotography were used to extract UnaG from our cells. Please note: The exact procedure can be found at the end of our UnaG wiki page. Conducting "bilirubin tests" (the addition of a small amount of bilirubin dissolved in chloroform to samples) allowed us to see if UnaG was present in our samples, since as mentioned earlier UnaG fluoresces in the presence of bilirubin.

Bilirubin test of cells before lysis.

• Lb + bilirubin/chloroform solution
• Lb culture of cells transformed with [[Part:BBa K2669000]] + bilirubin/chloroform solution
• LB culture of cells transformed with [[Part:BBa K2003011]] + bilirubin/chloroform solution
• LB culture of cells transformed with[[Part:BBa K2669000]] without bilirubin

Figure 2: Bilirubin test before/after affinity chromatography. Going from right to left the samples are:

• Lysed sample of the “bad” part before AC
• Lysed sample of the “good” part before AC
• "Bad" part after AC
• "Good" part after AC

Figure 3: Comparison of blank tube to successful extraction/previous iGEM part. The tubes reading from left to right are as followed:

• Blank tube with AC elution buffer/bilirubin
• Tube with bilirubin + original iGEM UnaG part
• Our extracted modified UnaG with a moved start codon, as can be seen in Figure 1

A good degree of fluorescence can be seen in the last tube compared to the other two, which clearly contain none of our protein of interest.

Figure 4: SDS-PAGE gel after affinity chromatography

UnaG is approximately 15.6 kDa, showing that it is indeed in the extracted sample. Other proteins are shown, and this is likely because we used no imidazole in the initial running buffer, leading to unspecific binding. We did this to ensure that we obtained as much UnaG as possible in our sample so that we could conduct fluorescence tests visible by the naked eye.

Figure 5: Fluorescence measurement of unlysed cells. From left to right: Bacterial strain BL21 transformed with a plasmid containing Part:BBa_K2669000 from 2018, Bl21 transfected with Part:BBa_K2003011 from 2016 and normal BL21 cells, all at roughly the same OD600 value.

Figure 6: The supernantant of lysed cells before and after the HisTrap chromotography. Because of our lysis method UnaG was suspended in the supernatant of the cell cultures. The left samples is supernantant containing the UnaG-protein from 2016 and the right samples are the supernantant containing our UnaG-protein (2018).

Conclusion

The histidine tag seems to function as intended. In addition, the amount of UnaG produced seems to be sufficient to both extract the protein of interest and to observe its florescence both when cells are lysed and when they are intact.

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