Title

Hanging off the Wall: Spider Protein Expression

Poster Number

14

Format

Poster Presentation

Faculty Mentor Name

Craig Vierra

Abstract/Artist Statement

Spider silk is one of the strongest natural materials found in nature. It is five times stronger than steel. The purpose of the study was to build a DNA construct that would allow the expression of a black widow spider silk gene in bacteria. Although the precise function of this gene is currently unknown, its expression pattern is restricted to silk-producing glands and its amino acid sequence has internal repeat blocks, a characteristic of silk family fibroin members. To generate the prokaryotic expression vector carrying a piece ofthis putative silk gene, polymerase chain reaction (PCR) was used to amplify a segment of this eDNA that encoded the C-terminal region of the putative silk gene. PCR products were monitored using agarose gel electrophoresis. Due to the repetitive nature of the sequence, multiple bands resulted during the PCR. A single band that had the closest expected size of 618 base pairs was extracted and purified from the agarose gel. Purified PCR products were inserted into the prokaryotic expression vector pBAD-TOPO and then transformed into competent E. coli cells. Restriction digestion analysis demonstrated that the expression plasmids carried the amplified cDNAs in the correct reading frame. We then attempted to overexpress this protein in bacteria. Our long-term goal is to use the purified protein to generate polyclonal antibodies in rabbits. These antibodies will then will be used further characterize the endogenous protein in the spider.

Location

Wendell Phillips Center, 1st floor hallways

Start Date

3-5-2008 1:00 PM

End Date

3-5-2008 3:00 PM

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May 3rd, 1:00 PM May 3rd, 3:00 PM

Hanging off the Wall: Spider Protein Expression

Wendell Phillips Center, 1st floor hallways

Spider silk is one of the strongest natural materials found in nature. It is five times stronger than steel. The purpose of the study was to build a DNA construct that would allow the expression of a black widow spider silk gene in bacteria. Although the precise function of this gene is currently unknown, its expression pattern is restricted to silk-producing glands and its amino acid sequence has internal repeat blocks, a characteristic of silk family fibroin members. To generate the prokaryotic expression vector carrying a piece ofthis putative silk gene, polymerase chain reaction (PCR) was used to amplify a segment of this eDNA that encoded the C-terminal region of the putative silk gene. PCR products were monitored using agarose gel electrophoresis. Due to the repetitive nature of the sequence, multiple bands resulted during the PCR. A single band that had the closest expected size of 618 base pairs was extracted and purified from the agarose gel. Purified PCR products were inserted into the prokaryotic expression vector pBAD-TOPO and then transformed into competent E. coli cells. Restriction digestion analysis demonstrated that the expression plasmids carried the amplified cDNAs in the correct reading frame. We then attempted to overexpress this protein in bacteria. Our long-term goal is to use the purified protein to generate polyclonal antibodies in rabbits. These antibodies will then will be used further characterize the endogenous protein in the spider.