Team:ZJUT-China -

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The aim of our project is to cut antibiotic resistance genes in engineering bacteria and reducing resistance genes in industrial emissions in industry. After ARGs are eliminated, cells are induced to lysin in our final design. In order to achieve this goal, we found the part BBa_K2277000 constructed by 2017 iGEM team ZJUT-China. In this part, the lysin gene is already in the plasmid. If you want to know about more details, You can read the paper “A light-controlled cell lysis system in bacteria” published in Journal of Industrial Microbiology & Biotechnology.

But in most cases, resistance genes are being inserted to plasmids for screening transformants. So we wanted to construct the lysin gene into the genome, in which way would have two advantages. The one is that it can reduce the number of plasmids that needed to be transformed into the engineering bacteria, thereby reducing the additional metabolic pressure. The other is that it could avoid the addition of new resistance. Thus, we submitted BBa_K2556051 as an improvement of BBa_K2277000. The design of this part is showed in Fig. 1.

Fig.1 BBa_K2556051

Our improvement include two aspects: 1) We improved the original basic part to a composite part, then future iGEM teams can use this part directly and use arabinose to regulate the expression of the lysin gene. 2) We added homological arms of E. coli genome to both ends of the part. If people want to integrate this part into the genome via CRISPR/Cas, they can directly use our part as donor DNA to facilitate their experiments. Furthermore, homological arms allowed us to insert this part into the non-metabolic pathway in E. coli genome, so inserting genes does not affect the normal growth of E. coli.

We tested BBa_K2277000 and BBa_K2556051 separately on plasmids and results are showed in Fig. 2. The original part and the improved part did not have many differences in lysis function, and the growth curve showed a significant decline after being induced.

Fig 2. Growth curve of E.coli with original part(PL)/improved part(TL)

Then we inserted our improved part into E. coli genome using CRISPR/Cas technology. In order to obtain transformants that were successfully inserted the part, we screened by plate streaking. The experimental results are showed in Fig 3.

Fig.3 Result of plate streaking

Finally, we selected 34 transformants, and 5 of them showed lysis effects on plates containing arabinose. They were probably the strains which had already inserted lysis part into genome. Unfortunately, we did not complete the growth curve test before wiki froze. However, we got a strain with significant lysis effect after arabinose induction, and we named it as: E. coli MG1655-Lysis. The result are showed in Fig. 4.

Fig.4 E.coli MG1655-Lysis cultured in tubes

Through our improvement, the arabinose-regulated lysin gene can be more easily integrated into the genome of E. coli. Thereby reducing the number of plasmids that needed to be transformed in bacteria can also reduce the additional metabolic pressure. What’s more, it can even reduce the resistance genes carried by bacteria which are potential factors for increasing bacterial resistance. We believe that this improvement described above is exactly meaningful.