Team:Uppsala/JoesPage

UnaG Troubleshooting

One of the biobrick parts submitted by the 2016 Uppsala team was UnaG combined with a histidine tag, for extraction in affinity chromatography. While observing this sequence however, we found that there was an error, and no histidine tag would express due to the start codon being placed after the histidine tag. In addition, this part would also express less or no UnaG at all due to the RBS now having a significant amount of space between it and the start codon. We decided to improve this biobrick part by moving the start codon to its proper location and then proving that the histidine tag works by extracting and purifying the protein via affinity chromatography.


However extraction of this protein poses some difficulty. UnaG, many other chromoproteins, is a membrane protein and therefore needs special conditions to purify. The following report will show how we successfully extracted and purified UnaG from BL21 E. coli cells expressing our custom made plasmid.

Design


Figure 1: Our annotated modified UnaG sequence with an N-terminal his tag. The terminator, RBS, and promoter sequences were all obtained from the iGEM website. The UnaG gene was taken from the iGEM website and only the start codon was moved so that the gene would properly express with a histidine tag. The start codon was previously immediately after the histidine tag. Note that two plasmids were designed, one using the original UnaG part from the iGEM 2016 Uppsala team and one modified one as shown above. The only modification between the two plasmids is the repositioned start codon.

Results

Cell lysis and affinity chromotography were used to extract UnaG from our cells. Bilirubin tests (addition of a small amount of bilirubin to samples) allowed us to see if the UnaG was present in our samples, since as mentioned earlier UnaG fluoresces in the presence of 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

UnaG can be observed in all tubes except the third one, which should not have a histidine tag since we used the 2016 part that was on the iGEM registry and therefore should not bind in the IMAC column. This supports our claim that our new part functions and provides a histidine tag to the protein, and the old part did not.


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.

Results 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.


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.

Digging Deeper

In order to visualize the intended reporter method in a more quantitative manner, we decided to use a plate reader to measure the intensity of various concentrations of GFP expressing cells in equine feces.

Samples of horse manure were mixed with water and GFP expressing cells into well plates. The absorbance was measured after this in order to see if it was possible to detect our chromoprotein in the mix.

We determined that at certain concentrations we could observe the presence of GFP vs none. This method is not as quantitative as we would like, but serves as a proof of concept for further experiments.