Difference between revisions of "Part:BBa K2808002"
 
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<partinfo>BBa_K2808002 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2808002 SequenceAndFeatures</partinfo>
  
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===Experimental Validation===
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Throughout our project, we successfully ligated our gene <i> invA </i> to the pSB1CR linearized backbone and transformed it into competent DH5-alpha <i>E. coli</i> cells. The part was validated by running a double restriction digestion reaction and PCR on the gene fragment using the respective PCR primers and the amplified regions were obtained as expected. Appropriate bands were seen in the respective lanes, which indicated that the part was successfully ligated and transformed.
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"https://static.igem.org/mediawiki/parts/thumb/e/e9/IGEM_NYU_Abu_Dhabi_2018_BioBricks_PCR_amplification.png/360px-IGEM_NYU_Abu_Dhabi_2018_BioBricks_PCR_amplification.png"
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"https://static.igem.org/mediawiki/parts/thumb/5/50/IGEM_NYU_Abu_Dhabi_2018_BioBricks_double_digestion.png/364px-IGEM_NYU_Abu_Dhabi_2018_BioBricks_double_digestion.png"
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===Source===
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Salmonella typhimurium
  
 
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<partinfo>BBa_K2808002 parameters</partinfo>
 
<partinfo>BBa_K2808002 parameters</partinfo>
 
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==Contribution by 2022 iGEM Team YkPaO==
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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.
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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.
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===Construction of the gene InvA containing plasmids===
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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.
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[[File:T--YkPaO--BBa K4304009-figure1.png|500px|thumb|center|Figure 1. the sequencing data of InvA DNA fragment.]]
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===Cleavage experiment - cleavage of oligo DNA===
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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).
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[[File:Contribution-fig2.png|500px|thumb|center|Figure 2.Gel electrophoresis comparing before and after cleavage of oligo DNA.
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M = Marker, NC = Negative Control.]]
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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.
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You can also see our information on the wiki page https://2022.igem.wiki/ykpao/contribution

Latest revision as of 09:29, 13 October 2022


Invasion protein (invA) gene for the detection of Salmonella typhimurium

The invA gene is involved in the invasion of the cells of the intestinal epithelium and could be involved in the translocation of the InvE protein in Salmonella typhimurium. Due its specificity to the organism and the lack of toxicity, various research projects have used this gene for the detection of S. typhimurium.

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 1123
    Illegal BglII site found at 1927
    Illegal BamHI site found at 1818
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 602
    Illegal NgoMIV site found at 1113
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1079
    Illegal SapI.rc site found at 1108

Experimental Validation

Throughout our project, we successfully ligated our gene invA to the pSB1CR linearized backbone and transformed it into competent DH5-alpha E. coli cells. The part was validated by running a double restriction digestion reaction and PCR on the gene fragment using the respective PCR primers and the amplified regions were obtained as expected. Appropriate bands were seen in the respective lanes, which indicated that the part was successfully ligated and transformed.


"360px-IGEM_NYU_Abu_Dhabi_2018_BioBricks_PCR_amplification.png"


"364px-IGEM_NYU_Abu_Dhabi_2018_BioBricks_double_digestion.png"


Source

Salmonella typhimurium


Contribution by 2022 iGEM Team YkPaO

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