Coding

Part:BBa_K2361000

Designed by: Mart Bartelds   Group: iGEM17_Groningen   (2017-10-09)


spdCas9

This part contains the protein coding sequence for the dCas9 originating from Streptococcus pyogenes. This protein is a catalytically-dead Cas9 variant, which lacks endonuclease activity. It is used for gene repression using CRISPR-interference. It can been seen as an improvment of part BBa_K1026001 since the illegal EcoRI site has been removed and the part was fully sequenced (see experience).


Usage and Biology

Overexpression of dCas9 may cause cytotoxic effects [1], therefore we recommend expressing it using an inducible promotor. In this part the start codon (ATG) starts directly behind the XbaI site and not at the last A of the XbaI site. This may lead to reduced efficiency of expression when combined with RBS's from the iGEM repository, but more importantly this needs to be taken into consideration when creating fusion proteins with this part to ensure that no frame-shifts occur.

References

[1] Nielsen, A. A., & Voigt, C. A. (2014). Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks. Molecular Systems Biology, 10(11), 763. http://doi.org/10.15252/msb.20145735

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1100
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 3379
    Illegal XhoI site found at 4115
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]
  • Experimental validation

    . This part was validated experimentally in the same experiment as dCas9VRER by iGEM Groningen 2017. For this experiment we have designed four gRNA's that target GFP. Two of this gRNA's bind to sequences flanked by NGG (1&2) and two bind to a sequence flanked by NGCG (3&4). These gRNA's were inserted into the plasmid pSB3C5 containing a pLacGFP construct as well. For spdCas9 and dCas9VRER expression those parts were put into a pBad vector in which they are expressed behind an Arabinose inducible promoter.

    In total 18 E. coli strains were produced containing pBad:dCas9, pBad:dCas9VRER or no pBad combined with pS3C5 with one of the gRNA's, pSB3C5 without a gRNA or no pSB3C5 (Table below).

    mindCas9dCas9VRER
    dH5αdCas9:nopSB3C5dCas9VRER
    noPbad:nogRNAdCas9:nogRNAVRER:nogRNA
    noPbad:gRNA1dCas9:gRNA1VRER:gRNA1
    noPbad:gRNA2dCas9:gRNA2VRER:gRNA2
    noPbad:gRNA3dCas9:gRNA3VRER:gRNA3
    noPbad:gRNA4dCas9:gRNA4VRER:gRNA4

    All strains were grown overnight and the next day they were dilute to an OD of 0.05 in the afternoon. At the end of the afternoon at ODof 0.4-0.6 all cultures were induced with a final concentration of 0.01% arabinose. All induced cultures were put back into the incubator (37C 220 rpm) and grown overnight. The next morning the OD of all cultures was measured and to an OD of 0.2 in LB (in final volume of 1mL). The OD and GFP (470/510) of all samples were measured in a plate reader in quadruplicate. The data obtained from the fluorescence measurement were converted to relative fluorescence and are shown in figure 5. To convert the fluorescence to relative fluorescence we first divided all measured fluorescence values were divided by the measured OD's. Next the fluorescence/OD of the negative controls (samples without pSB1C3) was subtracted from the other samples. Next all normalized fluorescence/OD values were divided by that of the positive control (no pBad:pSB3C5 without gRNA).

    Grabh.jpg

    From figure 5 it can be seen that all fluorescence values are quite similar for the samples without gRNA. Further we can see a clear decrease in the relative fluorescence of dCas9 in combination with gRNA 2 and 3. For gRNA's 1 and 4 the relative fluorescence of the dCas9 is similar to that of the samples without the pBad vector.

  • [edit]
    Categories
    //chassis/prokaryote/ecoli
    //function/crispr/cas9
    Parameters
    biology
    protein