Composite

Part:BBa_K5254018:Design

Designed by: Mana Hayashida   Group: iGEM24_Boston-BOSLab   (2024-09-27)


PCR fragment Chloramphenicol resistance gene flanked with 5’ and 3’ region of cysH gene from Nissle


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 41
    Illegal XhoI site found at 900
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Design

Fig 1: Knockout Cassette Design

Name: BBa_K5254003 Part type: Device Short description (PCR fragment Chloramphenicol resistance gene flanked with 5’ and 3’ region of cysH gene from Nissle 1917): Long description : Chloramphenicol resistance gene in ID42 parts from 2024 Distribution kit was amplified with primers directing 5’ end and 3’ end of CMR. These primers are also flanked by 40 nt from 5’ (3097442-3097403) and 3’ (3096628-3096667) UTR region of cysH gene (3096668-3097402) in Nissle 1917. PCR product should be CMR flanked with homologous arm to 5’ and 3’ UTR regions of the cysH gene (phosphoadenosine phosphosulfate reductase).


Figure 4. Recombination of the knockout cassette into cysH locus by lambda Red recombinase system

Design Notes

Upon testing our designs we noticed that the transformation of A1 and B1 into the CysH nissle knock out became increasingly difficult. It seems that cysH ko makes the Nissle resistant against the transformation by electroporation. We observed that chemical transformation was a better option for increasing transformation efficacy.

Because Nissle 1917 is not optimal for plasmid propagation, recovered plasmids from the Nissle have more endogenous digestion. Therefore, the plasmid confirmation by RE digestion can be challenging.

We could have designed FRT regions in our cassette to either remove the cmR resistance later or replace that gene with a modification enzyme. This would create an entirely in-vivo system.

Source

Joshi Lab - Lambda Red Recombinase

References

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