Coding

Part:BBa_K2351012

Designed by: Giel Scheepers   Group: iGEM17_Utrecht   (2017-10-23)


secreted Cpf1 - PhytoBrick

Please note: PhytoBrick in Universal Acceptor Plasmid (BBa_P10500)
Prefix fusionsite: 1234 = AATG // Suffix fusionsite: 5678 = GCTT
See BBa_K2351006 for illegal BioBrick version and BBa_K2351010 for codon-corrected BioBrick version.

AsCpf1 containing a signalsequence (Ig lambda-2 chain V region signal sequence) and histag (GGGHHHHHH). Codon optimized for human cell lines.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 248
    Illegal PstI site found at 1310
    Illegal PstI site found at 3719
    Illegal PstI site found at 3917
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 248
    Illegal PstI site found at 1310
    Illegal PstI site found at 3719
    Illegal PstI site found at 3917
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 432
    Illegal BglII site found at 1974
    Illegal BglII site found at 2283
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 248
    Illegal PstI site found at 1310
    Illegal PstI site found at 3719
    Illegal PstI site found at 3917
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 248
    Illegal PstI site found at 1310
    Illegal PstI site found at 3719
    Illegal PstI site found at 3917
    Illegal NgoMIV site found at 3518
  • 1000
    COMPATIBLE WITH RFC[1000]

Validation

The construct was sequenced to verify the correct insertion into the backbone.

The aim of the experiment was to secrete the protein into growth medium, using HEK293t cells.This was validated by showing the presence of Cpf1 in medium with Western Blots using Cpf1 antibodies.

Hek293t cells were transfected with ‘secreted Cpf1 plasmid’. After three days the medium and cells were harvested. A complete protocol can be found on our wiki: http://2017.igem.org/Team:Utrecht/secretion

Figure 1a shows the samples from HEK293t cell medium, lysed cells and protein purified from medium. Cpf1 is shown to be present in the medium, and could be further purified using Ni-purification. Figure 1b shows a Western Blot confirming the previous results. The thickness of the band for sCpf1 in cell medium, indicates that the product is indeed secreted protein and not the product of involuntary cell lysis.

Utrecht_sCpf1_registry.png

Figure 1: Western Blot

Rabbit Anti-Cpf1 was used as a primary antibody (1:2000), goat anti-rabbit-HRP was used as a secondary antibody (1:10 000). sCpf1 represents Cpf1 containing the signal sequence and histag.


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