Part:BBa_K4304009

invA Plasmid

invA Plasmid

Contribution

InvA, which is essential for Salmonella spp. to enter cultured epithelial cells, is a member of a family of proteins involved in either flagellar biosynthesis or the secretion of virulence determinants by a number of plant and mammalian pathogens. We designed this plasmid by inserting InvA 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 InvA containing plasmids

We send the company to synthesize the DNA fragments of InvA, 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 InvA 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).

Figure 2.Gel electrophoresis comparing before and after cleavage of oligo DNA. M = Marker, NC = Negative Control.

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
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1116
  Illegal BglII site found at 1920
  Illegal BamHI site found at 1811
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 595
  Illegal NgoMIV site found at 1106
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1072
  Illegal SapI.rc site found at 1101