Part:BBa_K4183004

Testing the synthesis efficiency of bacterial surface capsular polysaccharide (CAP).

CAP is a non-toxic podoconjugate polysaccharide derived from E. coli Nissle 1917 (EcN) for use in tumor therapy in vivo. This component was used to test the efficiency of CAP synthesis in EcN.

Usage and Biology

CAP is an EcN-derived non-toxic bacterial capsular polysaccharide that has been used in bacterial therapy for tumors in vivo. [2] It protects the bacterium from external environmental disturbances, such as extreme pH, enzymes, and other microorganisms. We hope to overexpress CAP on the surface of EcN to protect our engineered bacteria from being harmed by gastric acid. We designed it to survive in the complex flora of the intestine without affecting the original microenvironment of the intestine.

The synthesis of CAP requires the participation of various enzymes, such as kfiABCD, kpsCFSU and etc. Among them, kfiC is one of the key enzymes, and if the kfiC gene is overexpressed in the bacterium, CAP can be synthesized in large quantities.

We transferred the BBa_K4183004 plasmid into EcN and cultured it overnight in LB medium supplemented with IPTG. The CAP was subsequently purified by chloroform-phenol extraction, electrophoresed in SDS-PAGE (4%-20%), and stained using alcian solution. The results showed that an increased amount of CAP could be easily observed in the engineered bacteria overexpressing kfiC compared to the negative control (Figure 1).

Figure 1. SDS-PAGE verified the expression of CAP.

Since CAP improves bacterial tolerance to extreme PH environments in the stomach, we tested the engineered bacteria in different acidic environments.

The results showed (Figure 2) that the engineered bacteria overexpressing CAP could grow slowly in PH = 4 compared to the negative control. We also found that it could survive in the environment with PH ≤ 3 for about 30 min. After a person eats, the food will stay in the stomach for 1-1.5 h. This indicates that the engineered bacteria overexpressing CAP have some acid tolerance, but they cannot fully survive in the stomach acid environment.

Figure 2.The growth of bacteria in different PH environments, (A) is the negative control group and (B) is the experimental group.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 433
  Illegal PstI site found at 1565
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 433
  Illegal PstI site found at 1565
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 433
  Illegal BglII site found at 1015
  Illegal BamHI site found at 1368
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 433
  Illegal PstI site found at 1565
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 433
  Illegal PstI site found at 1565
  
• 1000
  COMPATIBLE WITH RFC[1000]