Multimerization of Block Repeats of MaSp1 Spider Silk Gene through Seamless Cloning Strategy for Expression Studies
Poster Number
38
Format
Poster Presentation
Faculty Mentor Name
Craig Vierra
Faculty Mentor Department
Biological Sciences
Abstract/Artist Statement
Western black widow spiders have seven types of silk glands that each produce a different silk fiber that serves distinct biological functions. Silk fibers possess tensile strength greater than steel and also exhibit high elasticity, leading to a tough material. Being able to synthetically produce silk fibers with the same levels of tensile strength, elasticity, and toughness could lead to many advancements in science and technology, such as strong and durable ropes and cables, medical sutures, string instruments, and replacements for Kevlar (body armor). Though there are many benefits, multiple challenges exist in the production of synthetic spider silk. One of the obstacles is expressing large amounts of the full length proteins in bacteria or yeast. Since the native silk proteins are over 300-kDa and have repetitive amino acid sequences, known as block repeats, investigators have struggled to synthesize recombinant proteins that have similar protein lengths. PCR amplification is also problematic, since the spider silk genes contain repetitive DNA sequences that are GC-rich. To circumvent these challenges, we devised a seamless cloning strategy that allows us to multimerize the MaSp1 block repeats to express a recombinant protein that has a molecular mass that approaches the native size of MaSp1 proteins. A synthetic 1x MaSp1 module was created by annealing oligonucleotides that were codon optimized for expression in bacteria. A Western blot was conducted to analyze the expression of the synthetic gene. Overall, this seamless cloning strategy can allow for multimerization of MaSp1 block repeats to express a recombinant protein whose molecular mass approaches the size of native MaSp1 proteins and could be used for many industrial applications.
Location
DeRosa University Center, Ballroom
Start Date
26-4-2014 2:00 PM
End Date
26-4-2014 4:00 PM
Multimerization of Block Repeats of MaSp1 Spider Silk Gene through Seamless Cloning Strategy for Expression Studies
DeRosa University Center, Ballroom
Western black widow spiders have seven types of silk glands that each produce a different silk fiber that serves distinct biological functions. Silk fibers possess tensile strength greater than steel and also exhibit high elasticity, leading to a tough material. Being able to synthetically produce silk fibers with the same levels of tensile strength, elasticity, and toughness could lead to many advancements in science and technology, such as strong and durable ropes and cables, medical sutures, string instruments, and replacements for Kevlar (body armor). Though there are many benefits, multiple challenges exist in the production of synthetic spider silk. One of the obstacles is expressing large amounts of the full length proteins in bacteria or yeast. Since the native silk proteins are over 300-kDa and have repetitive amino acid sequences, known as block repeats, investigators have struggled to synthesize recombinant proteins that have similar protein lengths. PCR amplification is also problematic, since the spider silk genes contain repetitive DNA sequences that are GC-rich. To circumvent these challenges, we devised a seamless cloning strategy that allows us to multimerize the MaSp1 block repeats to express a recombinant protein that has a molecular mass that approaches the native size of MaSp1 proteins. A synthetic 1x MaSp1 module was created by annealing oligonucleotides that were codon optimized for expression in bacteria. A Western blot was conducted to analyze the expression of the synthetic gene. Overall, this seamless cloning strategy can allow for multimerization of MaSp1 block repeats to express a recombinant protein whose molecular mass approaches the size of native MaSp1 proteins and could be used for many industrial applications.