Part:BBa_K4304008

ipaH Plasmid

ipaH Plasmid

Contribution

Shigella is a class of non-spore-forming Gram-negative pathogens whose pathogenicity depends on the secretion system. Shigella ipaH family proteins are co-encoded by virulence large plasmids and genes on chromosomes. We designed this plasmid by inserting ipaH DNA fragment into the plasmid pUC57 with a T7 promoter and terminator in front and after the sequence. This plasmid is used to obtain the gene fragment of Shigella which will bind with sgRNAs and recruit cas12a protein to cleave the DNA fragment of it.

Construction of the gene ipaH containing plasmids

We send the company to synthesize the DNA fragments of ipaH, and inserted it into the pUC57 vector, the sequenced data showed that the plasmids which were provided by the company were successfully constructed (Figure 1). The constructed plasmids were contained in E. coli strains, we streak inoculated them on LB solid medium plates containing corresponding antibiotics, and incubate them at 37℃ overnight, then we extracted the plasmids.

Figure 1. the sequencing data of ipaH DNA fragment.

Cleavage experiment - cleavage of oligo DNA

In order to verify if FnCas12a we purified could precisely recognize and cut the target DNA sequence, we developed an in vitro reaction platform. Firstly, we obtained the sgRNAs through an in vitro transcriptional method and extracted the target sgRNAs fragments. Next, we mixed the purified FnCas12a protein, the sgRNAs, the corresponding plasmids containing DNA fragments, and the reaction buffer together. Then we incubated the reaction system at 37°C for 2 hours, and we verified the result by gel electrophoresis (Figure 2).

insert figure 2

After Cleavage is constituted of oligo DNA after cleavage by cas12a protein and sgRNA. In contrast, NC (negative control) contains oligo DNA before cleavage only. Compared to NC, The oligo DNA band is significantly diminished after cleavage. This displays that the in vitro cutting experiment is successful.

You can also see our information on the wiki page https://2022.igem.wiki/ykpao/contribution

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

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  Illegal SapI.rc site found at 73
  Illegal SapI.rc site found at 964
  Illegal SapI.rc site found at 1552