E. coli is an important protein expression host strain, so it is important to develop expression plasmids. In order to distinguish the recombinant strain from other strains, the resistance genes play important roles in this process.
In the past iGEM teams, the iGEM16_Istanbul_Tech team provide a plasmid, pET28a (BBa_K2141000), which is a nonviral expression plasmid used in bacteria strains for protein production. It only has the kanamycin resistance gene as a selection marker. In our project, we inserted an Ampicillin resistance gene into the plasmid through an in vitro gene-editing system.
In order to amplify if the recombinant plasmid was successfully constructed, we coat the transformed competent cells on the double-antibiotics LB culture medium and calculated the number of colonies the next day. As a result, compared to the negative control, the transformed strain successfully grew on the double-antibiotics LB culture medium.

pET28a is one of the most commonly used expression vectors in E. coli. The vector is a high-copy-number plasmid. When expressed in the prokaryotic system, the Kanamycin resistance can be used to screen the right colony, and the strain should be cultured at 37℃. The protein expression can be induced by IPTG. This plasmid backbone can be used to express different proteins in the future.

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 1). 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 2).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 2).

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.

The casposon is a member of a distinct superfamily of archaeal and bacterial self-synthesizing transposons that employ a recombinase (casposase) homologous to the Cas1 endonuclease. It has a strong sequence preference in the presence of a proper target site, so we use the A. boonei casposase to catalyze casposons integration into specific regions.The casposon is a member of a distinct superfamily of archaeal and bacterial self-synthesizing transposons that employ a recombinase (casposase) homologous to the Cas1 endonuclease. It has a strong sequence preference in the presence of a proper target site, so we use the A. boonei casposase to catalyze casposons integration into specific regions.

Target site duplication (TSD) is a feature of Target-Primed Reverse Transcription (TPRT) that is necessary to consider when detecting novel insertions. The integration reaction appears to proceed via a two-step mechanism with casposons, whereby the first nucleophilic attack occurs at the TSD segment border by the 3′-OH of the spacer. After the formation of the half-site intermediate, the second nucleophilic attack occurs on the opposite strand at the junction between the TSD segment and the flanking DNA.

pRSFDeut-vector is one of the most commonly used expression vectors in E. coli and it is designed for the co-expression of two target genes with double expression promoters. The vector is a high-copy-number plasmid, and it contains the N-His flag. When expressed in the prokaryotic system, the Kanamycin resistance can be used to screen the right colony, and the strain should be cultured at 37℃. This plasmid backbone can be used to express different proteins in the future.

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