Team:CSWProteens/project

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Abstract: Annually, about 5% to 15% of agricultural produce is lost due to frost. The formation of ice damages plants by rupturing cells and also through dehydration as water molecules are drawn out of tissue. Current solutions to this problem - such as using heat or covering crops with protective material - are cumbersome, costly, and not fully preventative. Synthetic antifreeze chemicals have not been proven to work. Even if they did, they would be need to be applied repeatedly, at great cost, and may also leave residues in the environment.

The CSW ProTeens aim to design a synthetic biology solution to this problem with a nonpathogenic strain of E. coli able to produce and secrete RiAFP (Rhagium inquisitor Antifreeze Protein) using a part that the Yale ‘11 iGEM team developed. RiAFP is an insect antifreeze protein from Rhagium inquisitor and the most efficient AFP known. RiAFP, unlike synthetic chemicals, should not be harmful if ingested. We are not sure yet whether this method is effective on an industrial scale and will need to test this. We wish to use a type 1 secretion system designed by the Utah State University ‘09 iGEM team to transport the protein directly to the extracellular space. Plantifreeze is a dual plasmid system. Our device is designed to mitigate crop damage caused by ice crystal formation on the plant surface.

Plasmid #1. One plasmid controls RiAFP expression which is tagged with a 6x His motif and a HlyA secretion signal. RiAFP/6xHis/HlyA is a fusion protein. The second plasmid, pLG575, carries HlyBD proteins necessary for HlyA-tagged protein secretion. Initiation of transcription of RiAFP/6xHis/HlyA is controlled by a phage promoter, T7. A double terminator stops transcription. Thus, the genetic circuit contained in plasmid #1 is assembled from two parts and must adhere to the biofusion standard:

• Part A : 7T-RiAFP/His (no stop codon)

• Part B : HlyA/double terminator (no start codon)