Coding

Part:BBa_K4247010:Design

Designed by: Matteo Soana   Group: iGEM22_UCopenhagen   (2022-09-24)
Revision as of 12:28, 24 September 2022 by Ms1002 (Talk | contribs) (→‎Design considerations=)


Minispidroin_NT-2rep-CT_SnoopTag_N-6His


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 493
    Illegal PstI site found at 499
    Illegal PstI site found at 514
    Illegal PstI site found at 568
    Illegal PstI site found at 586
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 493
    Illegal PstI site found at 499
    Illegal PstI site found at 514
    Illegal PstI site found at 568
    Illegal PstI site found at 586
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 493
    Illegal PstI site found at 499
    Illegal PstI site found at 514
    Illegal PstI site found at 568
    Illegal PstI site found at 586
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 493
    Illegal PstI site found at 499
    Illegal PstI site found at 514
    Illegal PstI site found at 568
    Illegal PstI site found at 586
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The DNA sequence coding for the N- and C-terminus would be separated by a spacer containing a BsaI site and the repetitive part of the protein would have 2 BsaI sites on each end such that when Golden Gate Cloning was performed, the repetitive part would be inserted in between the N- and C-terminus to give the whole minispidroin protein. The DNA sequence coding for the minispidroin protein will be contained in a pET24 expression vector containing a T7 promoter, terminator and a 6x His-tag following the C-terminus of the protein to facilitate protein purification.


However, since the type IIS assembly compatibility system forbids the presence of a BsaI recognition site within the sequence of a part, we have chose to split the N- and C-terminus into 2 basic parts.


Further, this sequence has been codon optimised as per E.coli's codon bias.


It is difficult to synthesise the DNA sequence coding for the central part of the minispidroin due to its repetitiveness. So, at the UCopenhagen team, we have decided to split the protein into the N-terminus and C-terminus in one and the repetitive part in another plasmid. Using Golden Gate Assembly, the repetitive part can be inserted in between the N and C terminus to get the coding sequence of the entire minispidroin protein. In this way, any sequence can be added in between the N and C terminus to get a whole protein.


Since for some proteins, the expression is better with the His-tag on the N-terminus instead of the C-terminus we used primers to switch the 6x His-tag.


Further, we wanted to incorporate the SnoopTag with our protein such that it would enable the protein to bind to any other protein that has the complementary SnoopCatcher. The SnoopTag coding sequence was added to the C-terminus of the protein via PCR and User cloning.



Source

The sequence of this composite part is obtained from the following basic parts: BBa_K4247005 (Minispidroin_NT_N-6His), BBa_K4247001 (Minispidroin_2rep), BBa_K4247002 (Minispidroin_CT) and BBa_K4247008 (SnoopTag).


References