Using plasmids as vectors to introduce foreign genes into engineered bacteria is the most common approach in genetic engineering. But plasmids in microbiology is unstable, which is major caused by genetic instability and structural instability:

(1) Structural instability: Generally speaking, foreign genes for engineered bacteria are not necessary, which means when the foreign genes mutant and loss their function, the host can still survive, so the mutations would gradually accumulate and cause great harm to foreign genes. Meanwhile, because plasmids could exist in many forms, some host use homologous recombinases to integrate plasmids, resulting in damage to the plasmids’ structure.

(2) Genetic instability: when the engineered bacteria divide, there is usually no mechanism to guarantee the average distribution of plasmids, so foreign genes imported through plasmids do not have genetic stability.

The degradation of engineered bacteria is essentially caused by growth rate. Degraded strains have more growth advantages than the non-degraded ones. Because plasmids itself will bring additional metabolic burden for engineered bacteria or the expression of foreign genes have some toxicity, therefore the more copy of the plasmids or the more expression of the toxic protein, bacteria grow slower. On the contrary, the small copy number, no plasmid or plasmids loss of function, the bacteria’s growth rate is much higher than that of non-degraded strains. Eventually, in the whole population of bacteria, degradation strains accumulate more and more, and functional strains less and less.

Currently, the conventional method is to add an independently expressing resistant gene into the plasmids. The resistant gene can degrade a special kind of antibiotic, so the bacteria with plasmids can obtain resistance to the antibiotic. Bacteria with low or no copies of plasmids will be killed or suppressed when antibiotics were added into the culture, while strains with high copies retained. This method is widely used in the laboratory. However, there are some disadvantages:

(1) the problem of structural instability of plasmids cannot be solved. After the target gene loses its function, the resistant gene can still be expressed smoothly, which leads to that the bacteria screened by the resistant environment are not thoroughly.

(2) the application of this method is restricted. It could be used in the laboratory, but there would be a huge cost due to the using of antibiotics in industry;

(3) those bacteria with resistant genes will metabolize antibiotics, so the concentration of antibiotics in the culture would gradually reduce, which is not conducive to long-term experiments.

(4) adding antibiotics to the culture is exactly adding selection pressure, so engineered bacteria need to use a large part of energy to fight against those survival pressure, which leads to relatively low expression of target genes in engineered bacteria.

To sum up, the use of antibiotics in the construction, screening and application of engineered bacteria can NOT be avoided, but there are also many problems. In this summer, BNU-china are devoted to explore a novel method to fight against plasmids loss in the environment without antibiotics, and hope that our efforts could finally change the world.