UnaG Design

Below we present the changes (improvements i.e.) made to the pre-existing biobrick submitted by the 2016 Uppsala team together with a comparison of the results obtained from expressing and extracting the two UnaG variants. In short, we demonstrate how we obtained a fully working HIS-tag as well as a slightly higher level of expression of UnaG from plasmid.

Conclusion

With the above experiments, we have shown that we successfully modified the 2016 UnaG part to maintain proper functionality while adding in a constiative promoter + RBS + double terminator.

We have also shown that we have improved the “Inducible Green Fluorescent Protein UnaG+6xHis-tag+Flexible linker” protein part from the iGEM website by making it properly express a histidine tag that allows it to be extracted in affinity chromatography. This is shown in figures 3 and 4. In addition, we have also shown that we have an increased yield for UnaG than the previous iGEM part as can be seen in figure 5. Figure 6 shows that even using the plate reader there is little to no UnaG present in the 2016 sample after IMAC, which suggests a histidine tag was not expressing. The combination of fluorescence after IMAC purification and the correct sized band on the gel proves our biobrick part functions as intended. In addition, we developed a simple protocol to extract membrane proteins, which are traditionally notoriously difficult to extract.

The usage of the Triton X-100 incubation step theoretically created micelles in the solution, allowing membrane fragments to float around and protect UnaG since it is a beta barrel integral membrane protein that is quite hydrophobic in nature (Kumagai A, 2013). It was also found in the literature that using 5-10% glycerol [2] in all solutions involved in the extraction of integral membrane proteins is advised and theoretically helps keep them stabilized.

It may have been beneficial to express UnaG in a low copy plasmid, which might have lessened the risk that these heterologous proteins would conglomerate. We also chose to express UnaG in a constitutive manner, since we previously had no trouble expressing RFP or GFP (proteins with similar structures and environments) constitutively. This saved us time and also provided one less “moving part” that could go wrong in our experiment, such as an induction system not working properly.

UnaG Protocols

Transforming the Plasmid:

When the plasmids were received from IDT they were transformed into BL21 E. coli cells graciously provided to us by the Forster Laboratory. Same-day-made competent cells using the protocol "Making CaCl2 competent E.coli cells" 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” parts' 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.