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. The system utilizes the response of a protein with two catalytic domains which in cooperation catalyzes a reaction/process (e.g. syntheses of a compund or activation of transcription). Seperation of the two domains into individual peptides results in inactivation of the protein due to spatial seperation. The function can however be restored if the two domains are brought in close proximity, which can be accomplished by linking each domain to proteins or a protein and DNA that interacts with each other. Thus, coupling of the response resulting from the catalytic activity of the two domains with a reporter gene, enables for dertermination of protein-protein and protein-DNA interactions.
Our design is built 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 determine 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 at 4% that of monoclonal antibodies. Which leads us to conclude, that there are grounds to keep up the good work.