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Challenges in predicting the evolutionary maintenance of a phage transgene.

Schmerer M, Molineux IJ, Ally D, Tyerman J, Cecchini N, Bull JJ - J Biol Eng (2014)

Bottom Line: Consistent with the previous study, the dispersin phage was superior to unmodified phage at clearing short term biofilms grown in broth, shown here to be an effect attributable to free enzyme.There was little empirical support for the tragedy of the commons framework despite a strong theoretical foundation for its supposed relevance.Expressed from a different part of the genome, the transgene did behave as if intrinsically costly, but its maintenance did not benefit from spatially structured growth per se - violating the tragedy framework.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Computational Biology and Bioinformatics, The University of Texas at Austin, Austin, TX, USA.

ABSTRACT

Background: In prior work, a phage engineered with a biofilm-degrading enzyme (dispersin B) cleared artificial, short-term biofilms more fully than the phage lacking the enzyme. An unresolved question is whether the transgene will be lost or maintained during phage growth - its loss would limit the utility of the engineering. Broadly supported evolutionary theory suggests that transgenes will be lost through a 'tragedy of the commons' mechanism unless the ecology of growth in biofilms meets specific requirements. We test that theory here.

Results: Functional properties of the transgenic phage were identified. Consistent with the previous study, the dispersin phage was superior to unmodified phage at clearing short term biofilms grown in broth, shown here to be an effect attributable to free enzyme. However, the dispersin phage was only marginally better than control phages on short term biofilms in minimal media and was no better than control phages in clearing long term biofilms. There was little empirical support for the tragedy of the commons framework despite a strong theoretical foundation for its supposed relevance. The framework requires that the transgene imposes an intrinsic cost, yet the transgene was intrinsically neutral or beneficial when expressed from one part of the phage genome. Expressed from a different part of the genome, the transgene did behave as if intrinsically costly, but its maintenance did not benefit from spatially structured growth per se - violating the tragedy framework.

Conclusions: Overall, the transgene was beneficial under many conditions, but no insight to its maintenance was attributable to the established evolutionary framework. The failure likely resides in system details that would be used to parameterize the models. Our study cautions against naive applications of evolutionary theory to synthetic biology, even qualitatively.

No MeSH data available.


Related in: MedlinePlus

The dispersin transgene provides no benefit over control phages to the clearing of long term biofilms. Biofilm residue was measured with the crystal violet assay in biofilms that were grown 7 days in silicone tubing, then treated 4–5 days with phage (the leftmost bar is a no-phage control). T7+ and T7dsp (T7dsp+trx 10B) represent single-phage treatments, whereas T7v/T7dsp and T7+/T7dsp are from biofilms treated with equal mixes of control and transgenic phage. No significant heterogeneity is evident across the four phage-treated biofilms. Error bars represent 1 std. error; from left to right, the number of biofilms is 6, 6, 7, 2, and 2.
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Figure 5: The dispersin transgene provides no benefit over control phages to the clearing of long term biofilms. Biofilm residue was measured with the crystal violet assay in biofilms that were grown 7 days in silicone tubing, then treated 4–5 days with phage (the leftmost bar is a no-phage control). T7+ and T7dsp (T7dsp+trx 10B) represent single-phage treatments, whereas T7v/T7dsp and T7+/T7dsp are from biofilms treated with equal mixes of control and transgenic phage. No significant heterogeneity is evident across the four phage-treated biofilms. Error bars represent 1 std. error; from left to right, the number of biofilms is 6, 6, 7, 2, and 2.

Mentions: Biofilms were grown 7 days in silicone tubing at 37° in LB. This combination of time and media concentration enabled a robust biofilm to develop without complications of bacterial growth into the delivery or sampling tubes. In contrast to the short term biofilms, the week-old biofilms were visibly thick (several mm) but also irregular in macroscopic structure. They were also delicate in that their structure shifted if the tubing was physically disturbed.Pure phage populations or 1:1 mixtures of T7dsp and control phages were added on day 7. After 4–5 days of phage treatment, a CV assay was performed on the contents of the tubing (Figure 5). In contrast to the superiority of dispersin phages over control phages on short-term biofilms, all phages and phage combinations cleared the biofilm mass substantially, no differences being attributable to the transgenic dispersin. To ensure that dispersin activity had not been lost during these adaptations (which could explain the lack of a difference between dispersin-containing and control phages), dispersin activity of final phage populations was tested in short term biofilm assays; these tests were conducted with eluents of two long term biofilms that had been seeded with 100% T7dsp. Clearance of short term biofilms by these eluents was compatible with maintenance of functional dispersin (not shown). Thus the equivalence of the different phages on long term biofilms was not due to loss of dispersin functionality.


Challenges in predicting the evolutionary maintenance of a phage transgene.

Schmerer M, Molineux IJ, Ally D, Tyerman J, Cecchini N, Bull JJ - J Biol Eng (2014)

The dispersin transgene provides no benefit over control phages to the clearing of long term biofilms. Biofilm residue was measured with the crystal violet assay in biofilms that were grown 7 days in silicone tubing, then treated 4–5 days with phage (the leftmost bar is a no-phage control). T7+ and T7dsp (T7dsp+trx 10B) represent single-phage treatments, whereas T7v/T7dsp and T7+/T7dsp are from biofilms treated with equal mixes of control and transgenic phage. No significant heterogeneity is evident across the four phage-treated biofilms. Error bars represent 1 std. error; from left to right, the number of biofilms is 6, 6, 7, 2, and 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4128545&req=5

Figure 5: The dispersin transgene provides no benefit over control phages to the clearing of long term biofilms. Biofilm residue was measured with the crystal violet assay in biofilms that were grown 7 days in silicone tubing, then treated 4–5 days with phage (the leftmost bar is a no-phage control). T7+ and T7dsp (T7dsp+trx 10B) represent single-phage treatments, whereas T7v/T7dsp and T7+/T7dsp are from biofilms treated with equal mixes of control and transgenic phage. No significant heterogeneity is evident across the four phage-treated biofilms. Error bars represent 1 std. error; from left to right, the number of biofilms is 6, 6, 7, 2, and 2.
Mentions: Biofilms were grown 7 days in silicone tubing at 37° in LB. This combination of time and media concentration enabled a robust biofilm to develop without complications of bacterial growth into the delivery or sampling tubes. In contrast to the short term biofilms, the week-old biofilms were visibly thick (several mm) but also irregular in macroscopic structure. They were also delicate in that their structure shifted if the tubing was physically disturbed.Pure phage populations or 1:1 mixtures of T7dsp and control phages were added on day 7. After 4–5 days of phage treatment, a CV assay was performed on the contents of the tubing (Figure 5). In contrast to the superiority of dispersin phages over control phages on short-term biofilms, all phages and phage combinations cleared the biofilm mass substantially, no differences being attributable to the transgenic dispersin. To ensure that dispersin activity had not been lost during these adaptations (which could explain the lack of a difference between dispersin-containing and control phages), dispersin activity of final phage populations was tested in short term biofilm assays; these tests were conducted with eluents of two long term biofilms that had been seeded with 100% T7dsp. Clearance of short term biofilms by these eluents was compatible with maintenance of functional dispersin (not shown). Thus the equivalence of the different phages on long term biofilms was not due to loss of dispersin functionality.

Bottom Line: Consistent with the previous study, the dispersin phage was superior to unmodified phage at clearing short term biofilms grown in broth, shown here to be an effect attributable to free enzyme.There was little empirical support for the tragedy of the commons framework despite a strong theoretical foundation for its supposed relevance.Expressed from a different part of the genome, the transgene did behave as if intrinsically costly, but its maintenance did not benefit from spatially structured growth per se - violating the tragedy framework.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Computational Biology and Bioinformatics, The University of Texas at Austin, Austin, TX, USA.

ABSTRACT

Background: In prior work, a phage engineered with a biofilm-degrading enzyme (dispersin B) cleared artificial, short-term biofilms more fully than the phage lacking the enzyme. An unresolved question is whether the transgene will be lost or maintained during phage growth - its loss would limit the utility of the engineering. Broadly supported evolutionary theory suggests that transgenes will be lost through a 'tragedy of the commons' mechanism unless the ecology of growth in biofilms meets specific requirements. We test that theory here.

Results: Functional properties of the transgenic phage were identified. Consistent with the previous study, the dispersin phage was superior to unmodified phage at clearing short term biofilms grown in broth, shown here to be an effect attributable to free enzyme. However, the dispersin phage was only marginally better than control phages on short term biofilms in minimal media and was no better than control phages in clearing long term biofilms. There was little empirical support for the tragedy of the commons framework despite a strong theoretical foundation for its supposed relevance. The framework requires that the transgene imposes an intrinsic cost, yet the transgene was intrinsically neutral or beneficial when expressed from one part of the phage genome. Expressed from a different part of the genome, the transgene did behave as if intrinsically costly, but its maintenance did not benefit from spatially structured growth per se - violating the tragedy framework.

Conclusions: Overall, the transgene was beneficial under many conditions, but no insight to its maintenance was attributable to the established evolutionary framework. The failure likely resides in system details that would be used to parameterize the models. Our study cautions against naive applications of evolutionary theory to synthetic biology, even qualitatively.

No MeSH data available.


Related in: MedlinePlus