Difference between revisions of "Part:BBa K2027038"
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<partinfo>BBa_K2027038 parameters</partinfo>
 
<partinfo>BBa_K2027038 parameters</partinfo>
 
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===Characterization===
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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.
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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.
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https://static.igem.org/mediawiki/2016/1/14/T--Stanford-Brown--collagen_cpcr_gg2_s.png
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Figure: Gel electrophoresis results of CPCR of colonies transformed with digested constructs. There are 3 replicates picked from S1, S2, and S3 plates.

Revision as of 09:19, 24 October 2016


Bacterial Collagen with Coiled-Coil Heterotrimerization Domain 3

This part depends on parts BBa_K2027037 and BBa_K2027044 to function properly. See the design page or the relevant wiki[http://2016.igem.org/Team:Stanford-Brown/SB16_BioMembrane_Collagen] 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.