Team:University College London/Module 6

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Latest revision as of 02:50, 27 September 2012

Description

As our project suggests the release of genetically modified bacterium into the environment, we feel it is necessary to contain the risk of horizontal gene transfer. Horizontal gene transfer can occur by the release of genetic information through cell lysis, and subsequent transformation of other bacteria.

We decided we could target this system by constitutively expressing a nuclease. Any DNA released from the cell would therefore be digested, before it could diffuse, and transform wild-type bacteria. For this system we have selected a extracellular nuclease from Staphylococcus aureus (nucB), which has been well characterized (1-3) however it lacks the signal peptide for secretion to periplasm in E. coli. We chose DsbA (BBa_K243002) signal sequence that enables export of our nuclease to periplasm, thus it will allow us to digest extracellular genetic material.

As horizontal gene transfer can also occur via bacteria conjugation, we are proposing a multi-containment system, consisting of three toxin/anti-toxin pairs - Holin / Anti-Holin Endolysin, Colicin-E3 / Colicin Immunity E3, and Endunuclease EcoRI / Methyltransferase EcoRI.

Toxins will be carried on a plasmid and the antitoxin on the genomic DNA. As conjugation leads to the sharing of plasmid DNA, but not genomic DNA, the conjugating partner to our bacteria will receive the gene encoding the toxin, but not the related anti-toxin. Production of toxin in the absence of anti-toxin leads rapidly to cell death, which should prevent the sharing of genetic information.

Having such a system in place will prevent the transfer of synthetic genes into wild type bacteria; minimising the interference of our bacteria with the natural environment. Having more than one system is essential – even a system with 100% success at preventing horizontal gene transfer can be diminished by spontaneous mutations in the gene sequence. The use of separate systems is far more robust as it will provide reinforcement if a single system is knocked out. (Ronchel et al. 2001)

Reference

1. Okabayashi K, Mizuno D. (1974) Surface-bound nuclease of Stapylococcus aureus: purification and properties of the enzymes. Bacteriol. 117(1):222-6

2. Suciu D, Inouye M. (1996) The 19-residue pro-peptide of staphylococcal nuclease has a profound secretion-enhancing ability in Escherichia coli. Mol Microbiol. 21(1):181-95

3. Cooke GD, Cranenburgh RM, Hanak JA, Ward JM. (2003) A modified Escherichia coli protein production strain expressing staphylococcal nuclease, capable of auto-hydrolysing host nucleic acid. J Biotechnol. 101(3):229-39.

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-=Module 6: Containment=
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 == Description ==
-As our project suggests the release of genetically modified bacterium into the '''environment''', we feel it is necessary to '''contain''' the risk of s'''preading genetic information''' by '''horizontal gene transfer'''. To do so we are suggesting a '''trio''' of protective systems – consisting of two '''toxin/anti-toxin''' pairs  - Colicin-E3/Colicin Immunity E3 and Holin/Anti-Holin endolysin– and an '''excreted nuclease'''.
+As our project suggests the release of genetically modified bacterium into the '''environment''', we feel it is necessary to '''contain''' the risk of '''horizontal gene transfer'''. Horizontal gene transfer can  occur by the release of genetic information through cell lysis, and subsequent transformation of other bacteria.
+We decided we could target this system by constitutively expressing a nuclease. Any DNA '''released''' from the cell would therefore be '''digested''', before it could diffuse, and transform wild-type bacteria. For this system we have selected a extracellular nuclease from ''Staphylococcus aureus'' (nucB), which has been well  characterized (1-3) however it lacks the signal peptide for secretion to periplasm in ''E. coli''. We chose  DsbA (BBa_K243002) signal sequence that enables export of our nuclease to periplasm, thus it will allow us to digest extracellular genetic material.
-Having such a system in place will prevent '''transfer''' of '''synthetic genes''' into wild type bacteria; minimising the interference of our bacteria with the natural environment. Having more than one system is essential – even a system with 100% success at preventing HGT can be diminished by spontaneous mutations in the gene sequence. The use of '''three''' separate systems is far more '''robust''' as it will provide '''reinforcement''' if a single system is knocked out. (Ronchel et Ramos 2001)
+As horizontal gene transfer can also occur via bacteria conjugation, we are proposing a '''multi-containment''' system, consisting of three '''toxin/anti-toxin pairs''' - Holin / Anti-Holin Endolysin, Colicin-E3 / Colicin Immunity E3, and Endunuclease EcoRI / Methyltransferase EcoRI.
+Toxins will be carried on a plasmid and the antitoxin on the genomic DNA. As conjugation leads to the sharing of plasmid DNA, but not genomic DNA, the '''conjugating partner''' to our bacteria will receive the gene encoding the '''toxin''', but not the related '''anti-toxin'''. Production of toxin in the absence of anti-toxin leads rapidly to '''cell death''', which should prevent the sharing of genetic information.
-The system will be driven by a constitutive promoter BBa_J23119, to ensure that the release of genetic information is constantly neutralised.
+Having such a system in place will prevent the transfer of '''synthetic genes''' into wild type bacteria; minimising the interference of our bacteria with the natural environment. Having more than one system is essential – even a system with 100% success at preventing horizontal gene transfer can be diminished by spontaneous mutations in the gene sequence. The use of separate systems is far more '''robust''' as it will provide '''reinforcement''' if a single system is knocked out. (Ronchel ''et al.'' 2001)
+==Reference==
-Toxins will be carried on a plasmid and the antitoxin on the genomic DNA. As conjugation leads to the sharing of plasmid DNA, but not genomic DNA, the '''conjugating partner''' to our bacteria will receive the gene encoding the '''toxin''', but '''not''' the related '''antitoxin'''. Production of toxin in the absence of antitoxin leads rapidly to '''cell death''', which should prevent the sharing of genetic information.
+. Okabayashi K, Mizuno D. (1974) Surface-bound nuclease of Stapylococcus aureus: purification and properties of the enzymes. Bacteriol. 117(1):222-6
+. Suciu D, Inouye M. (1996) The 19-residue pro-peptide of staphylococcal nuclease has a profound secretion-enhancing ability in Escherichia coli. Mol Microbiol. 21(1):181-95
-'''Colicin E3''' is an antibiotic protein which has '''ribonuclease activity''' that targets the '''16S ribosomal RNA'''. This fatally impacts a cells ability to synthesise proteins, unless the '''antitoxin Im3''' is also present. Im3 is called an immunity protein – so named because it binds colicin E3 to prevent the ribonuclease activity. In our bacterial system, Colicin E3 will be present on a plasmid, and IM3 in the genomic DNA, so that both are '''constitutively''' produced. During conjugation, the conjugating partner will replicate, translate, and transcribe the Colicin E3 gene leading to '''cell death'''.
+. Cooke GD, Cranenburgh RM, Hanak JA, Ward JM. (2003) A modified Escherichia coli protein production strain expressing staphylococcal nuclease, capable of auto-hydrolysing host nucleic acid. J Biotechnol. 101(3):229-39.
-This will be reinforced by another toxin/antitoxin system –  a '''holin/antiholin endolysin pair'''. As before, the toxin '''holin''' will be carried by a plasmid and driven to constant expression by the constitutive promoter BBa_J23119. Holin causes '''disruption of the inner cellular membrane''', such that it allows an enzyme – '''endolysin''' – to enter the periplasm. Endolysin attacks the peptidoglycans of the periplasm, leading to '''cell lysis'''. Inhibition of this system can be achieved by having antiholin, which binds holin to prevent the disruption of the cell membrane, constitutively expressed in the genomic DNA. As before, conjugation with other bacteria will lead to replication of the damaging holin and endolysin genes, but not the rescue protein antiholin. All conjugating partners will suffer '''cell lysis''' as a result.
-However, HGT can also occur by the release of genetic information through cell lysis, and subsequent transformation of other bacteria.
-We decided we could target this system by '''constitutively''' expressing an extracellular nuclease. Any DNA released from the cell would therefore be digested, before it could diffuse. For this system we have selected a nuclease from staphylococcus aureus
-While the traditional concept of containment involves preventing biologicals from escaping into the environment, the advent of synthetic biology has causes a paradigm shift in this concept. As many synthetic biology projects target the environment, requiring the deliberate release of synthetic organisms into the biosphere, the concept of containment has to shift from 'preventing the release of synthetic organisms into the environment' to 'limiting the effects of synthetic organisms on the environment to their intended role'.
-Of course, this raises the question of whether the intended role of the synthetic organisms can themselves have any adverse effects on the environment. As such, the implementation of such systems goes beyond the role of science, to that of the society.
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