Composite
sN

Part:BBa_K2027007

Designed by: Charles Gleason   Group: iGEM16_Stanford-Brown   (2016-09-07)


Bacterial Collagen with Homotrimeric Coiled-Coil Domain

See the design page or the relevant wiki[1] for more details.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Characterization

After induction of our liquid cultures, we extracted the proteins from our constructs. We kept all protein washes to assure that our desired protein product did not get extracted earlier than expected (we expected the protein to be in the final elution buffer washes). After confirming its presence in the final elution washes (shown below), we ran the extracts against each other. To verify the size of the protein, we ran our protein extractions in SDS-PAGE gels. We mostly used Ni columns to purify our protein extract. Since our construct has a Lumio-Flag-His tag, we also used Anti-FLAG magnetic beads, with the help of our mentor Kosuke, to examine which protocol was most efficient in extracting protein.


T--Stanford-Brown--w123e123_c2cnsn.png

SDS-PAGE gel of all washes of extracted proteins using Ni-NTA resin columns and Lumio staining. AF stands for Anti-FLAG, and represents samples that were extracted using Anti-FLAG magnetic beads



The gel below also represents an SDS-PAGE gel containing the protein extracts of Part:BBa_K2027003 (natural construct without the elastin cross-linking domain or SN) in lanes 2-4 and Part:BBa_K2027002 in lanes 5-7. BBa_K2027002 is the fully assembled heterotrimeric coiled-coil collagen fiber monomer, consisting of the domains BBa_K2027004, BBa_K2027005, and BBa_K2027006. Because the fully assembled construct contains three domains, we expected the protein produced would be at least three times larger than the proteins with singular constructs. However, as indicated by the gel in Figure 2, BBa_K2027002 did not produce this size, which suggested that the assembly of all parts may have introduced errors in the protein synthesis.



T--Stanford-Brown--collagen_snfullc_page.png
SDS-PAGE gel of extracted SN and full construct proteins (lanes 2-4 and lanes 5-7 respectively) using Lumio stain

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