Composite

Part:BBa_K4687030:Design

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


MAD7+recE/T+pXMJ19+PlacM:MADE/TXlacM


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 9613
    Illegal XbaI site found at 9586
    Illegal SpeI site found at 5829
    Illegal SpeI site found at 6208
    Illegal PstI site found at 9574
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 9613
    Illegal SpeI site found at 5829
    Illegal SpeI site found at 6208
    Illegal PstI site found at 9574
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 9613
    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 BamHI site found at 9592
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 9613
    Illegal XbaI site found at 9586
    Illegal SpeI site found at 5829
    Illegal SpeI site found at 6208
    Illegal PstI site found at 9574
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 9613
    Illegal XbaI site found at 9586
    Illegal SpeI site found at 5829
    Illegal SpeI site found at 6208
    Illegal PstI site found at 9574
    Illegal NgoMIV site found at 7502
    Illegal AgeI site found at 1865
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

For gene editing using CRISPR technology, we need to transfect the plasmid into cells, allow it to express the CRISPR system and edit the target gene. To improve the efficiency of gene editing, we optimized the CRISPR system to determine the optimal CRISPR-Cas system.The CRISPR-MAD7 system initiates the genome editing process by creating targeted DNA breaks, while the recE/T system ensures the accurate and efficient integration of foreign DNA sequences into the bacterial genome at those breaks. And we have experimentally determined the optimal promoter PlacM. On this basis, we changed the vector skeleton to pXMJ19 to obtain the new recombinant plasmid, introduced it into Corynebacterium glutamicum, and observed the colony growth to determine the optimal plasmid skeleton.


Source

CRISPR-MAD7 nuclease was identified in Eubacterium rectale. RecE/T is derived from Bacterial,Archaeal and Plant Plastid Product. Strong promoter PlacM based on the sacB gene of Bacillus subtilis to be synthesized artificially. The vector skeleton pBluescript was E. coli-C. glutamicum shuttle expression vector. The main components in this recombinant plasmid are derived from the basic components: BBa_K4687000、BBa_K4687002、BBa_K4687008

References