Composite
s2

Part:BBa_K2027037

Designed by: Charles Gleason   Group: iGEM16_Stanford-Brown   (2016-10-08)


Bacterial Collagen with Coiled-Coil Heterotrimerization Domain 2

This part depends on parts Part:BBa_K2027044 and Part:BBa_K2027038 to function properly. 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
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 75
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 519
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 72
    Illegal BsaI.rc site found at 65


Characterization

We wanted to produce constructs without the elastin cross-linking domain. We removed this domain through Sap1 digestion of the DNA constructs and further transformed them into cells. We once again confirmed the success of the transformation with the digested constructs through gel electrophoresis and sequence verification. Although our sequence verifications were impressively similar to our desired constructs, it actually took 3 digestion attempts to fully digest the constructs to produce cells with constructs. While it is successful, the number of tries suggests that our Sap1 digestion with Golden Gate Assembly protocol could definitely be improved to increase efficiency.

We expect that digested constructs would be around 900 base pairs (bp). We used the gel to filter out picked colonies that did not contain the correct size of the constructs' DNA (such as S1.3). This was necessary, considering the inefficiency of the digestion protocol, so that we would only sequence colonies that were promising.

T--Stanford-Brown--collagen_cpcr_gg2_s.png


Figure: Gel electrophoresis results of CPCR of colonies transformed with digested constructs. There are 3 replicates picked from S1, S2, and S3 plates.


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


T--Stanford-Brown--collagenc2cn.png
SDS-PAGE gel of extracted C2 and CN proteins (lanes 2-5 and lanes 6-8 respectively) using Lumio stain. Lanes 2-5 represent the Lumio stained protein extract of the BioBrick Part:BBa_K2027005 or construct 2 (C2). Lanes 2,3, and 4 are respectively first, second, and third elution buffer washes using the His-tag nickel resin columns. Lane 5 is also C2's extracted protein, however it was extracted using a different protocol, Anti-FLAG. Lanes 6-8 contains corresponding elution buffers of the natural construct (CN), Part:BBa_K2027007. CN was extracted using His-tag nickel resin columns.

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