Title

Synthetic Production of MaSp1 Spider Silk Proteins via Seamless Cloning Strategy

Poster Number

39

Lead Author Major

Biological Sciences

Format

Poster Presentation

Faculty Mentor Name

Craig Vierra

Faculty Mentor Department

Biological Sciences

Abstract/Artist Statement

Spider dragline silk has been gaining interest from scientists across the globe due to its unique mechanical properties; it has a tensile strength stronger than steel and an extensibility that allows it to stretch to great lengths before breaking. The combination of the two traits allows the production of a tough material that can absorb much energy before failure, allowing it to be used in various medical and industrial applications. However, there is a limited amount of silk that a spider can produce, which makes farming for natural silk fibers impractical. Transformation and expression of spider silk genes in E. coli would allow for the mass production of synthetic fibers, but the native MaSp1 spider silk gene is too large to insert into a cloning vector or to express in bacteria. Because every organism has a different percentile of codons that codes for a certain protein, MaSp1 was codon optimized to create four oligonucleotides of GC-rich block repeats of the most used codons in E. coli for ideal bacterial expression. The oligonucleotides were annealed and the synthetic gene was amplified by PCR to create a 1x module insert for the vector. The pET24a vector underwent site-directed mutagenesis to mutate three unnecessary BsmB1 restriction sites that would have been problematic during a restriction digest. The single 1x module was inserted into the mutated pET24a expression vector, which was then transformed into E. coli cells to express the proteins necessary for silk production. Bacterial expression of the silk protein was confirmed through Western blot analysis. We plan on using the seamless cloning strategy to extend the 1x module insert to a 100x module of block repeats to create a synthetic gene that can produce a large enough protein to make synthetic fibers, bringing us a step closer to a mass production of synthetic spider fibers for real-world applications.

Location

DeRosa University Center, Ballroom

Start Date

26-4-2014 2:00 PM

End Date

26-4-2014 4:00 PM

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Apr 26th, 2:00 PM Apr 26th, 4:00 PM

Synthetic Production of MaSp1 Spider Silk Proteins via Seamless Cloning Strategy

DeRosa University Center, Ballroom

Spider dragline silk has been gaining interest from scientists across the globe due to its unique mechanical properties; it has a tensile strength stronger than steel and an extensibility that allows it to stretch to great lengths before breaking. The combination of the two traits allows the production of a tough material that can absorb much energy before failure, allowing it to be used in various medical and industrial applications. However, there is a limited amount of silk that a spider can produce, which makes farming for natural silk fibers impractical. Transformation and expression of spider silk genes in E. coli would allow for the mass production of synthetic fibers, but the native MaSp1 spider silk gene is too large to insert into a cloning vector or to express in bacteria. Because every organism has a different percentile of codons that codes for a certain protein, MaSp1 was codon optimized to create four oligonucleotides of GC-rich block repeats of the most used codons in E. coli for ideal bacterial expression. The oligonucleotides were annealed and the synthetic gene was amplified by PCR to create a 1x module insert for the vector. The pET24a vector underwent site-directed mutagenesis to mutate three unnecessary BsmB1 restriction sites that would have been problematic during a restriction digest. The single 1x module was inserted into the mutated pET24a expression vector, which was then transformed into E. coli cells to express the proteins necessary for silk production. Bacterial expression of the silk protein was confirmed through Western blot analysis. We plan on using the seamless cloning strategy to extend the 1x module insert to a 100x module of block repeats to create a synthetic gene that can produce a large enough protein to make synthetic fibers, bringing us a step closer to a mass production of synthetic spider fibers for real-world applications.