1 Providing more information on existing part (BBa_K322921)

We have compiled the information about sacB in the literature, such as the structure and function of the protein, to facilitate the follow-up investigation of this protein by other teams. More details can be seen on http://parts.igem.org/Part:BBa_K322921.

2 A CRISPR-based purification system "strainer" in E. coli for future teams

We developed a CRISPR-based purification system "strainer" in E. coli, which can remove the unsuccessfully edited cells, and then improve the overall editing efficiency. The "strainer" method utilized the double stranded DNA breaks (DSBs) as a signal to start the transcription of gRNA targeting on the plasmid harboring inducible toxic gene(sacB). Thus, the strain with successful recombineering can survive in the media with sucrose(without sacB gene). The strain without DSBs, still has sacB gene plasmid, cannot survive in the media with sucrose.

The previous studies have demonstrated that the SOS response is the first DNA repair system described in E. coli. The transcription of genes such as lexA, sula, umuD and recA are regulated by the SOS response. Thus, we can use the promoters of these genes to start the transcription of gRNAs targeting on the plasmid harboring a counter-selection markers if there is a double-stranded DNA breaks (DSBs) on the genome. When the sacB plasmid is cured by CRISPR/Cas system, the strain with successful recombineering can survival in the media with sucrose (Fig. 1). The strain without DSBs, still has the plasmid harboring sacB gene, cannot survival in the media with the sucrose. This is why we can increase the overall editing efficiency.

We firstly tested the new editing plasmid LexA using lexA gene promoter in EC85 to validate the "strainer" method. At a 0.01% sucrose concentration, the highest CFU/μg and the highest editing efficiency was found in the experiment. The editing efficiency using LexA plasmid is 25% higher than the control without "strainer" system. However, the CFU/μg decreased significantly compared to the control group that did not use "strainer". In addition, we used the 0.01% sucrose to tested another three new editing plasmids. All of them showed that the editing efficiency is 25% higher than the control without "strainer" system (Fig. 2A).

Firstly, the "strainer" system significantly improves the gene editing efficiency compared to the control without "strainer" system. Secondly, the CFU/μg is still 96% lower than that of the control even using 0.01% sucrose (Fig. 2B). To this end, we should further improve CFU/μg by designing and modifying SacB protein to reduce its toxicity.

More datils can be seen on https://2022.igem.wiki/dut-china/engineering.

3 Off-target predication model for future teams

Our off-target predication model can provide reference information for sgRNA usage. When people input the sgRNA sequences they designed and their target DNA sequences into the model, it can output off-target propensity information of these nucleotide sequences. This may help future teams to do such work. More details can be seen on https://2022.igem.wiki/dut-china/model.