Agarraberes, Fernando A.

Briggs, Samuel T

Oliveira, Paula F.

Weller, P. Tate

English

DigitalCommons@USU

From Spide to Bacte : Construction of Synthetic Genes of the Acinoform Spider Silk Protein (AcSp1)N-term  1     2     3      4     5    6      7     8     9    10   11   12   13  14  C-termAcSp1 geneHis tagAcSp1  8 repeatsAcSp1 10 repeatsAcSp1 12 repeatsAcSp1 14 repeatsFigure 2. A synthetic gene was designed containing two repeats of AcSp1. This gene was inserted in a plasmid that contains the ampicilin resistant gene (AMPr)1. This plasmid contains several restriction sites that disrupt the AMPr, the region encoding for the amino-terminal region and the carboxy-terminal region. This plasmid was used for the construction of all the AcSp1 genes. The ligation results in the restoration of the AMPr.P. Tate Weller, Samuel T. Briggs, Paula F. Oliveira, Fernando A. Agarraberes, and Randy V. LewisDepartment of Biology, Utah State University. Logan, UTSpider silks have remarkable physical properties due to a combination of strength and elasticity. In addition, spider silks are biocompatible and biodegradable. Our laboratory has shown that the strength products, such as fibers, produced with other silk proteins correlates with the size of the silk protein. The aciniform silk (AcSp1), has been shown to produce the thinnest and strongest fibers of all the natural spider silks. Aciniform silk is composed of a nonrepetitive amino-terminal region, 14 repeats of approximately 200 amino acids each, and a nonrepetitive carboxy-terminal region. We have been able to produce different versions of these genes encoding for 8, 10, 12, and 14 repeats. In addition, we were able to express these large proteins in E. coli. Figure 1. Schematic representation of AcSp1 genesAciniformglandORB WEAVING SPIDER SILKSORBIntroductionTechniques and StrategiesE. Coli electronmicrograph: http://www.explainthatstuff.com/electronmicroscopes.html1Hayashi CY, Blackledge TA, Lewis RV. Mol Biol Evol. 2004 Oct;21(10):1950-9. 3A 1      2      3     4      5bp20,00010,0007,000PlasmidmRNARibosomeAcSp1 (full length)AcSp1 (fragments)3BE. coli 20152575 50371501002503CkDaReferencesFigure 3. Plasmids containing different variants of AcSp1 and protein expression.(A) Constructs for different synthetic genes were selected by DNA size and sequenced. DNA ladder (lane 1), plasmid containing AcSp1 8 repeats (lane 2), 10 repeats (lane 3), 12 repeats (lane 4), and 14 repeats (lane 5). (B) Plasmids were introduced in the expression E. coli strain Bl21. ©. AcSp1 protein expression (Western blot analysis). Protein standards (lane 1), AsSp1 protein containing 8 repeats (lane 2), 10 repeats (lane 3), 12 repeats (lane 4), and 14 repeats (lane 5). Arrows indicate full length proteins.•Synthetic genes were successfully constructed•Genes are capable of expressing the corresponding proteins •Gene sequences need to be optimized to reduce/eliminate synthesis of protein fragmentsConclusions and Future WorkResultsOrb Weaving Spider Silks. The aciniform gland produces AcSp1 responsible for wrapping the prey and protecting the eggs. LigationRestriction Enzyme digestionsAcSp1 - 4 repeatsAcSp1 - 14 repeats     

Construction of Synthetic Genes of the Acinoform Spider Silk Protein (AcSp1)

https://digitalcommons.usu.edu/cgi/viewcontent.cgi?article=1019&amp;context=biology_posters

Construction of Synthetic Genes of the Acinoform Spider Silk Protein (AcSp1)

Abstract

Similar works

Full text

Available Versions

DigitalCommons@USU