Team:Caltech/Biosafety

General Concerns

There are a few risks in probiotic engineering that may prevent or delay our system from being implemented in humans. One common risk is increasing the chance of bacterial sepsis for those who have an immune deficiency. A few other risk factors include premature infants, CVC (central venous catheter), cardiac vascular disease, and diarrhea. ¹ We are also unsure of the effects that they could play a role in metabolic activities, and there could be unknown effects in implementing probiotics in humans. In addition, if we plan to supplement our strain with antibiotics for it to survive longer in the gut, such resistance may pass over into pathogenic strains which may cause future problems for the host. The concern of introducing foreign genes into the gut is described below for each individual project.

Subproject Concerns

Oxidative Burst

• Short Term: There are no safety concerns beyond that of typical E. coli lab strains. Considering that 260 mM H₂O₂ is applied directly to the skin to disinfect cuts and scrapes, the 800 uM H₂O₂ produced by the engineered strain should not be a health concern to anyone working in the lab.
• Long Term: Production of hydrogen peroxide is not a normal occurance in the large instestine, and its effects would need to be investigated before the engineered strain could be used to fight infection. Some concerns of hydrogen peroxide production in the intestine would be if:
  • it irritates the bowel².
  • it kills off significant amounts of the native gut flora.
  • it significantly damages gut epithelial cells.
  • the ability to produce peroxide can be transmitted to other gut flora.

Phage Pathogen Defense

Lactose Intolerance

• Synthetic LacY/Z Plasmid There are no known safety concerns regarding this plasmid. The only foreign enzyme not present in humans that is expressed is Beta-Galactosidase, which simply acts as lactase.
• Lysis Cassette Plasmid This plasmid may cause problems in the gut, if passed over from our strain to another strain. If passed over to a pathogenic strain, this may of course help the host in lysing that strain when lactose is present, but if passed over to a helpful strain, this may wipe out a great amount of our gut flora.

Vitamin Production

Population Variation

• The current constructs consist of GFP behind promoters. GFP is not known to be hazardous. However, in the simple fimE constructs, when the promoter starts in the configuration pointing upstream, DNA upstream of the constructs may be transcribed by the cell.
• In the final design, the terminator that sits in the population variation generator may not be 100% efficient. Thus, the efficiency of the terminator should be tested before genes that are hazardous when co-expressed are placed into the system.

References

1. Boyle RJ, Robins-Browne RM, Tang ML. Probiotic use in clinical practice: what are the risks? Am J Clin Nutr 2006;83:1256-64.
2. Ackermann M, Stecher B, Freed NE, Songhet P, Hardt WD, and Doebeli M. Self-destructive cooperation mediated by phenotypic noise. Nature 2008 Aug 21; 454(7207) 987-90.