• 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 the same OD600 value.

Figure 6: The supernantant of lysed cells before and after affinity chromotography. Because of our lysis method UnaG was suspended in the supernatant of the cell cultures. The left samples are 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.

Procedure:

Transforming the Plasmid:

When the plasmids were received from IDT they were transformed into BL21 E. coli cells graciously provided to us by the esteemed Forster Laboratory. Same-day-made competent cells using the protocol from the “Synthetic Biology Handbook” were used to provide maximum transformation efficiency.

Extraction of UnaG:

The protocol for the extraction of our integral membrane protein from the transformed BL21 cells proceeded as follows: Note that this was done for both iGEM 2016 cells transformed with the previous part (nicknamed “bad”) and our repositioned start codon (graced with the moniker “good”).

Materials/Procedure

• Lysis Buffer: PBS solution with 1mM EDTA, 5% glycerol, and 20mM Tris, pH7.4
• Elution Buffer: 20 mM sodium phosphate, 0.5 M NaCl, 0.5 M imidazole, pH 7.4, 5% glycerol PBS, 1mM EDTA, 5% glycerol, 20mM Tris, pH7.4
• Binding/Washing Buffer:0.5 M NaCl, 2 EDTA-free tablets, 10 % glycerol, 20mM sodium phosphate, 1% Triton x100, pH 7.4 (400 mL total)
• Binding/washing buffer with 1% triton x-100 by weight

Cells were centrifuged at 4000 g 25 minutes at 4 degrees Celsius and then resuspended in Lysis buffer. Cells were lysed using cell disruption with a french press. The now lysed cells were then centrifuged again at at 4000 g 25 minutes at 4 degrees Celsius. The pellet was resuspended in 20mL binding/washing buffer with 1% triton x-100. The solution was incubated on ice for one hour before another round of centrifugation at the same temperature and speed. After centrifugation the supernatant should contain the protein of interest. Bilirubin tests were conducted on both solutions of the pellet and supernatant to observe any fluorescence under a UV light.

Affinity chromatography was then performed on both “good” and “bad” solutions using prepacked “His-Gravitrap” Columns from GE Healthcare. The protocol for use was performed according to GE healthcare’s specifications, with modified binding/washing/elution buffers. After affinity chromatography, the resulting elutants were tested for fluorescence with a bilirubin test.

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