Coding

Part:BBa_K1763442

Designed by: Vinson Lam   Group: iGEM15_UCLA   (2015-09-15)
Revision as of 21:54, 18 September 2015 by Vinsonclam (Talk | contribs)

MaSp1/2[1:2]-12(1C3)

This is a 3-mer construct of MaSp2 constructed using Iterative Capped Assembly (ICA) protocols as outlined on the 2015 UCLA iGEM page [http://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/Protocols here]. This part is a coding sequence, with no regulatory elements present. This construct includes an N-terminal 8-his tag which can be used purify this protein using nickel based affinity purification techniques.

The construct has a ratio of 1 MaSp1 to 2 MaSp2 arranged in block style.

Biology

Native spider silk genes consist of many repeats of the MaSp genes, up to 100 repeats. The extensive repetition confers the properties of strength and elasticity that are commonly associated with spider silk threads. In order to examine the role of protein length and composition on the final properties of spider silk, we created this construct using Iterative Capped Assembly.


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 1264
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage

In order to express this construct as a protein, it is necessary to subclone it after a promoter and RBS. The 2015 UCLA iGEM team has made a composite part, BBa_K1763424, has placed this part under control of the T7-RBS regulatory elements.

This construct was successfully amplified using the [http://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/ICA_Oligo_Sequences post-elution primers] after ICA. The results of the amplification are shown below.

Fig. 1 XbaI and PstI digestion of all constructs made this summer. M1/2[1:2]-12 is at the very right. The expected product size is 1324 bp.


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Parameters
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