Coding

Part:BBa_K1085012

Designed by: Claudio Tiecher   Group: iGEM13_Groningen   (2013-09-10)

RBS Start-codon EstA Strep-tag SpiderSilkSubunitN1

This BioBrick contains the coding sequence for the protein EstA fused to the coding sequence of a novel spider silk protein (SubunitN1), with the Strep-tag in front of the latter. The construct also shows a Bacillus subtilis ribosome binding site (RBS) together with the starting codon (ATG).

The RBS is there to allow the ribosome to bind and start to translate the DNA.

The estA codes for the EstA precursor protein which therefore facilitates the secretion of the produced protein.

The Strep-tag, fused at the N terminal, is used both as a binding tag to use to indicate its production and as a binding component to Streptavidin.

SubunitN1 codes for a spider silk protein that is optimized for maximal expression in Bacillus subtilis 168. The primary goal of the algorithm was to optimize the codon selection based on their availability scores. The secondary goal was to prevent the formation of secondary RNA structures in close proximity to the RBS, and the tertiary goal was to minimize the number of restriction sites.

The amino acid sequence is based on the adapted MaSp2 sequences described in the paper of Brooks et. al. 2008.

Related Biobricks

BBa_K1085013 is the same except it has no Strep-tag between the EstA and SpiderSilkSubunitN1.

BBa_K1085010 is the same except it has a SpiderSilkSubunitE1 instead of a SpiderSilkSubunitN1.

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 650
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 707
    Illegal AgeI site found at 262
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//chassis/prokaryote/bsubtilis
//collections/silk
Parameters
None