Part:BBa_K2669000

Strongly constitutive His-tagged+flexible linker UnaG

UnaG is a unique chromoprotein since it expresses fluorescent signal when in contact with bilirubin. This means (unlike most other chromoproteins) it can be used as a reporter in anaerobic environments or even potentially in environments where bilirubin is naturally present, such as the intestines. While researching the previous work from the iGEM Uppsala 2016 team, we noticed that their part (Part:BBa_K2003011) had an error in it (a misplaced start codon) which would lead to not only reduced UnaG expression but also not include the histidine tag their part should be expressing according to the igem registry!

We decided that we would rectify this issue by engineering a composite part with the start codon in the correct position and compare it to the previous part (Part:BBa_K2003011). We designed this composite part by combining a strong promoter (Part:BBa_J23119), a strong RBS (Part:BBa_J34801), and a double terminator (Part:BBa_B0014) around the modified UnaG part (Part:BBa_K2669001).

Warning:This part also contains a GSG linker that has a stop codon directly after it. If you would like to use Part:BBa_K2669001 as a linker protein, you must delete or move this stop codon.

Source

As mentioned above, this part contains a strong promoter (BBa_J23119), a strong RBS (BBa_J34801), and a double terminator (BBa_B0014). In addition, it contains the sequence from BBa_K2003011, only with a change to the start codon location, resulting in Part:BBa_K2669001. The original source of UnaG is from the paper “A Bilirubin-Inducible Fluorescent Protein from Eel Muscle” by Kumagai A et. al, which characterized the UnaG protein from the muscle of a species of Japanese eel.

How we made it

Please note: The exact procedure can be found at the end of our UnaG wiki page.

iGEM Uppsala 2018 Experience + Experimental Data

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.

Figure 1: 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 2: 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 3: 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 4: 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.

Usage and Biology

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]