The Expression of ECP-1 Truncated in Bacteria
Poster Number
13
Format
Poster Presentation
Abstract/Artist Statement
Spider silk has extraordinary tensile strength and elasticity. Little is known regarding the molecular constituents that comprise egg case silk. In previous studies Egg Case Protein-1 (ECP- 1) was established to be predominantly produced in the tubuliform gland, with lower levels detected in the major and minor ampullate glands In this study, we expressed full-length ECP-1, as well as truncated versions of ECP-1 (N-terminal and C-terminal) using the prokaryotic expression vector pBAD. This study was performed in preparation for future experiments to be conducted this summer. This summer we want to determine if ECP-1 interacts with TuSp1 and if it does, determine the location of the interaction. TuSp1 is also found in the large diameter fibers of the egg case silk. Primers were selected to clone ECP-1 through PCR. Specific restriction sites were added to the 5’ termini of the primers to help determine directionality once the fragments were ligated into the plasmid. Following PCR, ECP-1 cDNA fragments were ligated into pBAD and transformed into bacteria. Restriction digests were used to determine the directionality of the fragments. Once the fragments were determined to be in the correct direction, a pilot expression was performed, followed by a western blot analysis. Through the analysis of the western blot it was determined that full length ECP-1 could be expressed, however, the expression was significantly reduced in comparison to the truncated versions. These results indicate that expression of full-length ECP-1 is likely toxic to the cells.
Location
Pacific Geosciences Center
Start Date
5-5-2007 1:00 PM
End Date
5-5-2007 3:00 PM
The Expression of ECP-1 Truncated in Bacteria
Pacific Geosciences Center
Spider silk has extraordinary tensile strength and elasticity. Little is known regarding the molecular constituents that comprise egg case silk. In previous studies Egg Case Protein-1 (ECP- 1) was established to be predominantly produced in the tubuliform gland, with lower levels detected in the major and minor ampullate glands In this study, we expressed full-length ECP-1, as well as truncated versions of ECP-1 (N-terminal and C-terminal) using the prokaryotic expression vector pBAD. This study was performed in preparation for future experiments to be conducted this summer. This summer we want to determine if ECP-1 interacts with TuSp1 and if it does, determine the location of the interaction. TuSp1 is also found in the large diameter fibers of the egg case silk. Primers were selected to clone ECP-1 through PCR. Specific restriction sites were added to the 5’ termini of the primers to help determine directionality once the fragments were ligated into the plasmid. Following PCR, ECP-1 cDNA fragments were ligated into pBAD and transformed into bacteria. Restriction digests were used to determine the directionality of the fragments. Once the fragments were determined to be in the correct direction, a pilot expression was performed, followed by a western blot analysis. Through the analysis of the western blot it was determined that full length ECP-1 could be expressed, however, the expression was significantly reduced in comparison to the truncated versions. These results indicate that expression of full-length ECP-1 is likely toxic to the cells.