Part:BBa_K3781209:Design
L1_sAP_RBD_mCerulean_GST, MocloMania Composite
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 598
Illegal PstI site found at 2101 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 598
Illegal PstI site found at 2101 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 536
Illegal XhoI site found at 6 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 598
Illegal PstI site found at 2101 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 598
Illegal PstI site found at 2101
Illegal NgoMIV site found at 684
Illegal NgoMIV site found at 1432 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 708
Illegal SapI.rc site found at 2073
Illegal SapI.rc site found at 2145
Design Notes
This composite part was assembled from four basic parts that are part of the MocloMania collection. Due to the highly standardizes set of overhangs that all our basic parts comply with, assembly could be achieved by a simple Moclo ligation. Assembling this part was of great importance to our project's prospect, since our objective was to test our expression system by expressing, secreting and purifying recombinant SARS-CoV2-RBD.
Having the fusion protein secreted into the cell's exterior, the culture medium, allows for the RBD to undergo glycosylation while passing the secretory pathway. Since facilitating the production of complexly glycosylated proteins is a core motivation in our project, generating and testing L1 constructs like this is very beneficial to achieving our research goal. Furthermore, assembly of this part allowed us to verify the correct construction of its sub-parts.
Introduction of a fluorescent tag such as mCerulean allows for easy protein detection in the downstream activity assays conducted which examine the functional binding of the recombinant RBD to ACE2 receptors overexpressed in a transgenic human cell line.
Source
For information on the origin of the genetic sequences present in this composite part, please consult the individual basic part pages.