In this project, we developed a precise gene-editing system in E. coli named casposons (Figure 1), in order to test the length of the integrated gene, we chose kanamycin as our reporter system.

In order to insert the TIR sequence into the pUC19 plasmid (Figure 2A), we use PCR to amplify the target DNA fragment and inserted the amplicons into DH10b competent cells. Inoculated a single colony into LB (Amp) culture medium, extracted the plasmid, and send to the company for Sanger sequencing (Figure 2B). As shown in the sequencing result, we successfully constructed the plasmid.

In order to obtain our target genes, we amplified different lengths of the target genes containing the Kanamycin gene fragment from the pUC19-DONER plasmid (Figure 3A). In order to successfully amplify the genes, we use different annealing temperatures, such as 59℃, 61℃, and 63℃ (Figure 3B).

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, add isopropanol into the reaction system and discard the supernatant, and resuspend the pellet with sterile water.

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 4).

Because the colonies on the plate are too intensive to calculate, we resuspended the colonies with LB culture medium, incubated at 37℃ for 30min, diluted them 8 times, coated them on the LB (Kana+Amp) solid medium plates and incubated them at 37℃ overnight (Figure 5). The next day, we calculated 1/4 area of the plate of the number of colonies (Figure 6). Because the colonies on the plate are too intensive to calculate, we resuspended the colonies with LB culture medium, incubated at 37℃ for 30min, diluted them 8 times, coated them on the LB (Kana+Amp) solid medium plates and incubated them at 37℃ overnight (Figure 5). The next day, we calculated 1/4 area of the plate of the number of colonies (Figure 6).

As a result, we can find that when the length of inserted gene is around 3.5k, we still achieved gene-editing with casposons. What’s more, as the length of the inserted gene increased, the number of colonies decreased. However, casposons is still an excellent tool we could use in future research for gene editing.

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 7.), and the plasmids were successfully constructed. So far, we have successfully developed our gene editing system.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 7.), and the plasmids were successfully constructed. So far, we have successfully developed our gene editing system.

We amplified the Ampicillin DNA fragment and promoter from template DNA and extracted it from the gel, and stored it at -20℃ for future use. In order to successfully amplify the genes, we use different annealing temperatures, such as 57℃ and59℃ (Figure 8). And as shown in the figure, there were two clear bands at 1kb can be seen, which means we correctly amplified the target gene fragment.

Mixed components according to the table below, reacted at 37℃ for 1h in the metal bath; 4uL PK enzyme (PK/ 0.5m EDTA V/V =1:1) was added to each tube, and digested in the metal bath at 37℃ for 30min and inactivated at 95℃ for 10min, added isopropanol into the reaction system and discard the supernatant, and resuspend the pellet with sterile water.

We transformed the recycled plasmids pool into E. coli DH10b competent cells, and coat on the LB solid medium plate containing both Kanamycin, Ampicillin antibiotics, and IPTG, incubated at 37℃ overnight. The next day, we calculated the number of colonies (Figure 9).

From the result, compared with the negative control, we can find that with casposase in the reaction system we successfully inserted the Ampicillin gene into the pET28a plasmid so that the strain could grow on the plate.

Wang X, Yuan Q, Zhang W, Ji S, Lv Y, Ren K, Lu M, Xiao Y. Sequence specific integration by the family 1 casposase from Candidatus Nitrosopumilus koreensis AR1. Nucleic Acids Res. 2021 Sep 27;49(17):9938-9952. doi: 10.1093/nar/gkab725. PMID: 34428286; PMCID: PMC8464041.