Composite

Part:BBa_K4687035:Design

Designed by: Yiming Jiang   Group: iGEM23_HBUT-China   (2023-10-07)


MAD7+recE/T+pJYS1+PlacM:MADE/TJlacM-g4


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 9481
    Illegal EcoRI site found at 9724
    Illegal EcoRI site found at 10600
    Illegal XbaI site found at 8698
    Illegal SpeI site found at 6464
    Illegal SpeI site found at 14418
    Illegal SpeI site found at 14797
    Illegal PstI site found at 7654
    Illegal PstI site found at 8944
    Illegal PstI site found at 9211
    Illegal PstI site found at 10405
    Illegal PstI site found at 12574
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 9481
    Illegal EcoRI site found at 9724
    Illegal EcoRI site found at 10600
    Illegal SpeI site found at 6464
    Illegal SpeI site found at 14418
    Illegal SpeI site found at 14797
    Illegal PstI site found at 7654
    Illegal PstI site found at 8944
    Illegal PstI site found at 9211
    Illegal PstI site found at 10405
    Illegal PstI site found at 12574
    Illegal NotI site found at 9811
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 9481
    Illegal EcoRI site found at 9724
    Illegal EcoRI site found at 10600
    Illegal BglII site found at 714
    Illegal BglII site found at 762
    Illegal BglII site found at 1083
    Illegal BglII site found at 2298
    Illegal BglII site found at 2950
    Illegal BglII site found at 3804
    Illegal BglII site found at 8507
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 9481
    Illegal EcoRI site found at 9724
    Illegal EcoRI site found at 10600
    Illegal XbaI site found at 8698
    Illegal SpeI site found at 6464
    Illegal SpeI site found at 14418
    Illegal SpeI site found at 14797
    Illegal PstI site found at 7654
    Illegal PstI site found at 8944
    Illegal PstI site found at 9211
    Illegal PstI site found at 10405
    Illegal PstI site found at 12574
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 9481
    Illegal EcoRI site found at 9724
    Illegal EcoRI site found at 10600
    Illegal XbaI site found at 8698
    Illegal SpeI site found at 6464
    Illegal SpeI site found at 14418
    Illegal SpeI site found at 14797
    Illegal PstI site found at 7654
    Illegal PstI site found at 8944
    Illegal PstI site found at 9211
    Illegal PstI site found at 10405
    Illegal PstI site found at 12574
    Illegal NgoMIV site found at 5134
    Illegal NgoMIV site found at 13112
    Illegal AgeI site found at 1865
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Traditional gene editing methods are not very efficient in Corynebacterium glutamicum. In order to improve the efficiency of gene editing in Corynebacterium glutamicum, we tried to construct a new system that can be expressed in Corynebacterium glutamicum that can perform gene editing efficiently.Whereas CRISPR-MAD7 nuclease has been described to target a wider range of PAM sequences, 5'-YTTN-3' , and exhibit high gene editing activity in microbial systems with small molecular weights, MAD7 can be used for a variety of gene editing. It has been reported that cas9 expression may retard the growth of Corynebacterium glutamicum due to toxicity of the gene product.CRISPR-MAD7 showed low toxicity in Corynebacterium glutamicum cells. Compared with the CRISPR-Cas9 system, CRISPR-MAD7 has a shorter crRNA length and a simpler secondary structure, which makes it more economical and convenient in designing, synthesizing, and transcribing crRNAs. After the optimization of CRISPR-MAD7 system in the experimental process, it can make its gene editing efficiency in Corynebacterium glutamicum reach 100% which is unattainable by other systems.Therefore, we hope to improve the gene editing efficiency in Corynebacterium glutamicum by optimizing the CRISPR-MAD7 system. For our subsequent knockdown of genes in Corynebacterium glutamicum.


Source

CRISPR-MAD7 nuclease was identified in Eubacterium rectale. RecE/T is derived from Bacterial,Archaeal and Plant Plastid Product.The PlacM promoter was synthesized artificially based on the Bacillus subtilis sacB gene.The vector skeleton pJYS1 was derived from the constitutive expression of FnCpf1 and recT in the C.glutamitum.The gRNAs are mainly designed based on the gene sequence of the PAM site. The sequence design mainly relies on the neighboring motifs located in the sequence of the pre-interval region of the PAM sequence (Protospacer Adjacent Motif).

References