Team:SDU-Denmark/Tour30
"Success is a science; if you have the conditions, you get the result." - Oscar Wilde
PAST
Two-hybrid screening is a way to determine protein-protein interactions or protein-DNA interactions. This system works by linking the proteins or DNA to each half of a transcription factor. The given transcription factor/or messenger protein is cut into 2 pieces. This leads to that the only way the transcription factor can relay a signal is of the 2 halves are connected, or brought in close proximity. The system is designed so that if the 2 proteins interact, this will bring together the 2 parts of the transcription factor/messenger protein and the signal will be relayed.
Our design is build around the catalytic activity of the adenylate cyclase in E. coli, which produces cyclic adenosine monophosphat (cAMP) from ATP in the bacteria. The catalytic domain of adenylate cyclase can be divided into 2 fragments; T18 and T25. When these 2 subunits come together it enables the production of cAMP. cAMP levels can be monitored by a cAMP-induced promoter expressing a reporter gene; in our case, Red Fluorescent Protein (RFP). RFP can be detected either by eye or quantitativly by flourecense microscopy. The variable part of the peptide aptamer will be generated through a nucleotide library. The library consist of different sequences generated from 60 random nucleotide basepairs flanked by a suffix and prefix, this can be considered the variable part of the peptide aptamer. This is inserted in a scaffold protein, Human Thioredoxin (hTrx), which can be considered as the constant part of the peptide aptamer. If the amino acid sequence of the variable part matches the target protein, RFP will be expressed in the cells and we have created a peptide aptamer for the given target protein.
Evaluation of large-scale production will be performed to determined whether it is valid to continue the work after iGEM. A production of 250 [batches/yr] of approximately one liter per batch with a concentration of 2 [g/L] is assumed appropriate on basis of a market analysis. A cost analysis was conducted and showed a profitability in all parameters at a price of 4% that of monoclonal antibodies. Which leads us to conclude, that there are grounds to keep up the good work.