Potential CEST Contrast Agents

Protein production is induced by the FNR promoter. Each of the 8 proteins listed below can be expressed once a hypoxic environment is sensed. These proteins are all rich in Arginine and Lysine amino acids, which are known to be good for CEST imaging.

The proteins that the CEST protein production device can generate are:

• Human Protomine 1 Optimized ([http://parts.igem.org/Part:BBa_K1123013 BBa_K1123013])
• 1ETF ([http://parts.igem.org/Part:BBa_K1123014 BBa_K1123014])
• 1PJN ([http://parts.igem.org/Part:BBa_K1123021 BBa_K1123015])
• 1G70 ([http://parts.igem.org/Part:BBa_K1123016 BBa_K1123016])
• Poly Arginine-Glycine ([http://parts.igem.org/Part:BBa_K1123018 BBa_K1123018])
• Poly Arginine-Serine ([http://parts.igem.org/Part:BBa_K1123019 BBa_K1123019])
• Poly Threonine-Lysine ([http://parts.igem.org/Part:BBa_K1123020 BBa_K1123020])
• Poly Lysine-Serine ([http://parts.igem.org/Part:BBa_K1123021 BBa_K1123021])

The first four proteins were selected using the RCSB Protein Data Bank. A python program was designed by us alongside a molecular dynamics simulator, to ensure that the selected proteins had a high content of Arginine and Lysine amino acids. The complete description of the protein selection process can be found on the protein selection section of the drylab tab. The rest of the proteins were selected using the results of McMahon's research (2008) [1].

These protein sequences were transformed into pET28a vectors for aerobic expression. When the proteins were over expressed and when the cell pellets were fixated in 4% PFA (paraformaldehyde) for two days, MRI CEST measurements were performed and the data was processed. For more detailed information about the lab procedures and the steps that were taken to come to these results, see CEST Agent Testing, for the results of the data processing, see MRI data processing.

References

[1] McMahon, M.T., 2008, New “Multi-Color” Polypeptide DIACEST Contrast Agents for MR Imaging, Magn Reson Med. 2008 October ; 60(4): 803–812.