Analyzing the Efficiency of Inserting GP10 into a Planar Bilayer Membrane Dopped with a Carboxylated Lipid

The phi29 connector, GP10, is a channel protein from the bacteriophage phi29, a virus that can infect bacteria. The protein provides a channel that allows the phi29 genomic DNA to go through it during the infection process in-vivo. It has been successfully demonstrated that the connector can be inserted into a planar lipid bilayer membrane in-vitro, and thus the channel protein could potentially be used as a single-molecule platform to detect individual molecules (both small molecules and macromolecules), to monitor chemical and biochemical reactions in real time, and to perform DNA/RNA sequencing. Understanding the process of inserting the protein translocation the planar bilayer membranes will allow scientists to design a platform for the novel biosensor. However, the current problem encountered is the difficulty in achieving stable and efficient insertions of the protein into a synthetic lipid, 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). For this purpose, in the research, we used molecular biology methods to prepare the protein, GP10 and employed a new strategy to optimize used lipids for efficient orthogonal integration of the phi29 connector into bilayer lipid membranes. Utilizing the precise knowledge of the 3-D structure of the phi29 connector, the bilayer membrane is optimized by tuning reactivity in the bilayer using the DPhPC doped with a carboxylated lipid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (PAcPC). The addition of the lipid into the bilayer would expectedly result in electrostatic attractive forces between the exposed basic amino acids on the surface of stalk in the phi29 connector and tails of the carboxylated lipid molecules in the core of the bilayer membrane. We will compare differences between the three particular factors that the data would give: the insertion efficiency, which is the total amount of insertions per sample time; the time of each insertion; and the total amount of insertions per sample batch. Furthermore, we will employ an independent two-tailed t-test to determine whether the differences are significant, thereby implying if the modified carboxylated lipid molecule does indeed stabilize the channel.http://opus.ipfw.edu/stu_symp2014/1055/thumbnail.jp