Part:BBa_K2027038
Bacterial Collagen with Coiled-Coil Heterotrimerization Domain 3
This part depends on parts Part:BBa_K2027037 and Part: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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 75
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 519
- 1000INCOMPATIBLE 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.
Figure: Gel electrophoresis results of CPCR of colonies transformed with digested constructs. There are 3 replicates picked from S1, S2, and S3 plates.
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