Team:Caltech/Biosafety

From 2008.igem.org

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==General Concerns==
==General Concerns==
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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. <sup>1</sup> We are also unsure of the effects that probiotics have on metabolic activities. 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.
+
There are 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 are immune deficient. Other risk factors include premature infants, CVC (central venous catheter), cardiac vascular disease, and diarrhea<sup>1</sup>. We are also unsure of the effects that probiotics have on host metabolic activities. However, the unmodified Nissle 1917 strain has been shown to be safe for long term use<sup>2</sup>.
 +
 
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The engineered probiotic will persist in the gut longer if antibiotics are used to suppress other gut flora<sup>3</sup>. However, such resistance may pass over into pathogenic strains and cause future problems for the host. The concern of introducing foreign genes into the gut is described below for each individual project.
==Subproject Concerns==
==Subproject Concerns==
===Oxidative Burst===
===Oxidative Burst===
*'''Short Term:''' There are no safety concerns beyond that of typical ''E. coli'' lab strains. Considering that 260 mM H<sub>2</sub>O<sub>2</sub> is applied directly to the skin to disinfect cuts and scrapes, the 800 uM H<sub>2</sub>O<sub>2</sub> produced by the engineered strain should not be a health concern to anyone working in the lab.
*'''Short Term:''' There are no safety concerns beyond that of typical ''E. coli'' lab strains. Considering that 260 mM H<sub>2</sub>O<sub>2</sub> is applied directly to the skin to disinfect cuts and scrapes, the 800 uM H<sub>2</sub>O<sub>2</sub> 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:
+
*'''Long Term:''' Production of hydrogen peroxide is not a normal occurrence in the large intestine, 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 bowel<sup>2</sup>.
+
**it irritates the bowel<sup>4</sup>.
**it kills off significant amounts of the native gut flora.
**it kills off significant amounts of the native gut flora.
**it significantly damages gut epithelial cells.
**it significantly damages gut epithelial cells.
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===Lactose Intolerance===
===Lactose Intolerance===
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* '''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.
+
* '''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 β-Galactosidase, which simply acts as a lactase. Several lactase preparations are formally 'Generally Recognized as Safe' by the FDA[http://www.cfsan.fda.gov/~rdb/opa-g132.html] and we expect no difficulties in finding a suitable 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.
* '''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.
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===Population Variation===
===Population Variation===
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* 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.
+
* The current constructs are regulatory in nature and present few safety concerns aside from those involved in the treatment subprojects. Minor concerns are discussed below:
 +
* 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.
* 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.
 +
* The engineered FimE system may interfere with natural fimbriae expression in Nissle 1917 or other gut flora. Since fimbriae are important for intestinal colonization, this could effect the intestinal composition.
==References==
==References==
# Boyle RJ, Robins-Browne RM, Tang ML. Probiotic use in clinical practice: what are the risks? Am J Clin Nutr 2006;83:1256-64.
# Boyle RJ, Robins-Browne RM, Tang ML. Probiotic use in clinical practice: what are the risks? Am J Clin Nutr 2006;83:1256-64.
 +
# Westendorf AM, Gunzer F, Deppenmeier S, Tapadar D, Hunger JK, Schmidt MA, Buer J, and Bruder D. '''Intestinal immunity of Escherichia coli NISSLE 1917: a safe carrier for therapeutic molecules'''. ''FEMS Immunol Med Microbiol'' 2005 Mar 1; 43(3) 373-84.
 +
# Rao S, Hu S, McHugh L, Lueders K, Henry K, Zhao Q, Fekete RA, Kar S, Adhya S, and Hamer DH. '''Toward a live microbial microbicide for HIV: commensal bacteria secreting an HIV fusion inhibitor peptide'''. ''Proc Natl Acad Sci U S A'' 2005 Aug 23; 102(34) 11993-8.
# 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.  
# 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.  
}}
}}

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Biosafety Concerns


General Concerns

There are 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 are immune deficient. Other risk factors include premature infants, CVC (central venous catheter), cardiac vascular disease, and diarrhea1. We are also unsure of the effects that probiotics have on host metabolic activities. However, the unmodified Nissle 1917 strain has been shown to be safe for long term use2.

The engineered probiotic will persist in the gut longer if antibiotics are used to suppress other gut flora3. However, such resistance may pass over into pathogenic strains and 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 occurrence in the large intestine, 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 bowel4.
    • 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 β-Galactosidase, which simply acts as a lactase. Several lactase preparations are formally 'Generally Recognized as Safe' by the FDA[http://www.cfsan.fda.gov/~rdb/opa-g132.html] and we expect no difficulties in finding a suitable 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 are regulatory in nature and present few safety concerns aside from those involved in the treatment subprojects. Minor concerns are discussed below:
  • 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.
  • The engineered FimE system may interfere with natural fimbriae expression in Nissle 1917 or other gut flora. Since fimbriae are important for intestinal colonization, this could effect the intestinal composition.

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. Westendorf AM, Gunzer F, Deppenmeier S, Tapadar D, Hunger JK, Schmidt MA, Buer J, and Bruder D. Intestinal immunity of Escherichia coli NISSLE 1917: a safe carrier for therapeutic molecules. FEMS Immunol Med Microbiol 2005 Mar 1; 43(3) 373-84.
  3. Rao S, Hu S, McHugh L, Lueders K, Henry K, Zhao Q, Fekete RA, Kar S, Adhya S, and Hamer DH. Toward a live microbial microbicide for HIV: commensal bacteria secreting an HIV fusion inhibitor peptide. Proc Natl Acad Sci U S A 2005 Aug 23; 102(34) 11993-8.
  4. 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.
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