Difference between revisions of "Part:BBa K4304011"

Revision as of 12:48, 26 September 2022

16s Plasmid

Contribution

the 16S DNA fragment is amplified from H.pylori genomic DNA. 16S ribosomal RNA sequences have been used extensively in the classification and identification of Bacteria and Archaea. The comparison of almost complete 16S rRNA gene sequences has been widely used to establish taxonomic relationships between prokaryotic strains, with 98.65% similarity currently recognized as the cutoff for delineating species. The DNA sequence of 16S is conserved, so it could also be used to distinguish H.pylori from other pathogenic microorganisms. This plasmid is used to obtain the 16S DNA fragment which will bind with sgRNAs and recruit cas12a protein to cleave the DNA fragment of it.

Figure 1. Plasmid 1: pUC57-16S plasmid containing strain.

Engineering Success

Construction of pathogenic micro-organisms expression plasmids

In order to construct our plasmids, we let the company synthesize the DNA fragments, the fragments 16S and cagA were inserted into the pUC57 vector. 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 (Figure 1).

Extraction of oligo DNA in plasmid

Figure 2. 1.5% TAE agarose gel electrophoresis to verify the construction of oligo DNA containing plasmids.

We used TAE agarose gel electrophoresis to testify the presence of oligo DNA in the plasmid by performing PCR and then doing gel electrophoresis of the amplicons (Figure 2).

Our results show that a band of 16S, but not in negative control (NC) lanes. Because oligo DNA has a size between 200bp to 400bp, our result supports the fact that the plasmids contain desired oligo DNA. The four plasmid transformations were successful.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 612
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 7: / Line 7: @@
 the 16S DNA fragment is amplified from H.pylori genomic DNA. 16S ribosomal RNA sequences have been used extensively in the classification and identification of Bacteria and Archaea. The comparison of almost complete 16S rRNA gene sequences has been widely used to establish taxonomic relationships between prokaryotic strains, with 98.65% similarity currently recognized as the cutoff for delineating species. The DNA sequence of 16S is conserved, so it could also be used to distinguish H.pylori from other pathogenic microorganisms. This plasmid is used to obtain the 16S DNA fragment which will bind with sgRNAs and recruit cas12a protein to cleave the DNA fragment of it.
-We used TAE agarose gel electrophoresis to testify the presence of oligo DNA in the plasmid by performing PCR and then doing gel electrophoresis of the amplicons (Figure 2).
-Our results show that a band of 16S, but not in negative control (NC) lanes. Because oligo DNA has a size between 200bp to 400bp, our result supports the fact that the plasmids contain desired oligo DNA. The four plasmid transformations were successful.
 [[File:T--YkPaO--BBa K4304011-figure1.png|500px|thumb|center|Figure 1. Plasmid 1: pUC57-16S plasmid containing strain.]]
@@ Line 19: / Line 17: @@
 We used TAE agarose gel electrophoresis to testify the presence of oligo DNA in the plasmid by performing PCR and then doing gel electrophoresis of the amplicons (Figure 2).
 Our results show that a band of 16S, but not in negative control (NC) lanes. Because oligo DNA has a size between 200bp to 400bp, our result supports the fact that the plasmids contain desired oligo DNA. The four plasmid transformations were successful.