Part:BBa_K4411021

TIR-1258bp Kan donor-TIR

TIR-1258bp Kan donor-TIR

Profile

Name: TIR-1258bp Kan donor-TIR

Base Pairs: 1258 bp

Origin: pET28a plasmid, TIR is synthesized

Properties: prokaryotes resistance gene fragment fused with TIR DNA fragment

Usage and Biology

This part is a composite part containing the Kanamycin gene fragment ad the TIR DNA fragment. Kanamycin is an inhibitor of protein biosynthesis that causes mRNA code misreading by binding to the 30S ribosome so that it could be used in E. coli host strains to screen for correct colonies. What’s more, TIR (Terminal Inverted Repeats) is the that could be recognized by the casposons and formed a complex with it. Then the TIR containing DNA fragment would be inserted into the TSD sequence and complete the gene editing [1-5].

Construct design

The TIR-1258bp Kan donor -TIR DNA fragment is amplified by PCR and was used in the in vitro reaction system (Figure 1).

Figure 1. the schematic diagram of the composite part BBa_K4411021..

Experimental approach

1. TIR-1258bp Kan donor-TIR Electrophoresis

Figure 2. The PCR gel electrophoresis result of the Kana gene fragment. M. DNA marker, 1. The annealing temperature is 59℃, 2. The annealing temperature is 61℃, 3. 1. The annealing temperature is 63℃.

In order to obtain our target genes, we amplified the target gene TIR-1258bp Kan donor-TIR from the kanamycin-containing plasmid. To successfully amplify the genes, we use different annealing temperatures, such as 59℃, 61℃, and 63℃ (Figure 2). In the figure, a clear and single DNA band at 1kp can be seen, indicating that we successfully amplified our target genes. We extracted the DNA fragments and stored them at -20℃ for future use.

2. In vitro casposons gene-editing system

To verify whether the long fragment gene with TIR sequence could be inserted into the TSD sequence effectively and correctly, the protein casposase was added for reaction, and the reaction products were recovered. Mixed components according to the table below, reacted in a metal bath at 37°C for 1h. Add PK enzyme at 37°C for 30min, then 95°C for 10min to terminate the reaction, added isopropanol into the reaction system and discard the supernatant, and resuspend the pellet with sterile water.

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3. Screen for TIR-Kan plasmids

We transformed the recycled plasmids pool into E. coli DH10b competent cells, and coat on the LB solid medium plate containing both Kanamycin and Ampicillin antibiotics, incubated at 37℃ overnight. The next day, we calculated the number of colonies on the plate (Figure 3).

Figure 3. The plates of recombinant plasmids containing strains. NC: pUC19-TSD, PC: pUC19 (Amp plate), 1258bp: PUC19-TSD-1258bp-TIR-Kan gene..

4. Sanger sequencing to amplify the recombinant plasmids We inoculate the single colony in the LB liquid culture medium (Kana+Amp), extracted plasmids, amplified the target-gene-containing fragments, and send the company for Sanger sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure 4), and the plasmids were successfully constructed. So far, we have successfully developed our gene editing system.

Figure 4. The sequencing data mapped to the plasmid sequence..

As the result shown above, compared with the negative control, we can find that with the casposase in the reaction system we successfully inserted the Kanamycin gene into the pUC19 plasmid so that the strain could grow on the plate containing antibiotics. So that we successfully developed a precise gene editing system in vitro, and this system could be used in future use or even be applied in clinical treatment.

References

1.Hickman AB, Dyda F. The casposon-encoded Cas1 protein from Aciduliprofundum boonei is a DNA integrase that generates target site duplications. Nucleic Acids Res. 2015 Dec 15;43(22):10576-87. doi: 10.1093/nar/gkv1180. PMID: 26573596

2.Krupovic M, Shmakov S, Makarova KS, Forterre P, Koonin EV. Recent Mobility of Casposons, Self-Synthesizing Transposons at the Origin of the CRISPR-Cas Immunity. Genome Biol Evol. 2016 Jan 13;8(2):375-86. doi:10.1093/gbe/evw006. PMID: 26764427; PMCID: PMC4779613.

3.Béguin P, Charpin N, Koonin EV, Forterre P, Krupovic M. Casposon integration shows strong target site preference and recapitulates protospacer integration by CRISPR-Cas systems. Nucleic Acids Res. 2016 Dec 1;44(21):10367-10376. doi: 10.1093/nar/gkw821. PMID: 27655632; PMCID: PMC5137440.

4.Krupovic M, Béguin P, Koonin EV. Casposons: mobile genetic elements that gave rise to the CRISPR-Cas adaptation machinery. Curr Opin Microbiol. 2017 Aug; 38:36-43. doi: 10.1016/j.mib.2017.04.004. PMID: 28472712; PMCID: PMC5665730.

5.Béguin P, Chekli Y, Sezonov G, Forterre P, Krupovic M. Sequence motifs recognized by the casposon integrase of Aciduliprofundum boonei. Nucleic Acids Res. 2019 Jul 9;47(12):6386-6395.doi:10.1093/nar/gkz447.PMID:31114911; PMCID: PMC6614799.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 138
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 211
  Illegal SapI site found at 1231