Difference between revisions of "Team:UMaryland/Measurement"

 
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PET degradation has been and is a <a href="https://2018.igem.org/Team:UMaryland/PETaseIntro"><u>popular iGEM project</u></a>. However, measurement of this degradation remains a challenge.
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PET degradation has been and is a <a href="https://2018.igem.org/Team:UMaryland/History"><u>popular iGEM project</u></a>. However, measurement of this degradation remains a challenge.
 
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<img src="https://static.igem.org/mediawiki/2018/6/6f/T--UMaryland--PNPBissue.png" style="height: inherit; width: inherit;" alt="Waluigi Time!">
 
<img src="https://static.igem.org/mediawiki/2018/6/6f/T--UMaryland--PNPBissue.png" style="height: inherit; width: inherit;" alt="Waluigi Time!">
 
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Figure from Yoshida et. al.
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Figure from Yoshida et. al. [1]
 
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Despite being much more effective at degradation of PET, PETase produces minimal results in a PNPB absorbance assay.</div>
 
Despite being much more effective at degradation of PET, PETase produces minimal results in a PNPB absorbance assay.</div>
 
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Measurement of PET degradation remains a challenge for three major reasons.
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Measurement of PET degradation is difficult for three major reasons.
 
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First, the fastest PET degrading enzyme currently on the registry, PETase, is highly specific for PET. This means that despite its superior degrading ability, it will actually produce weaker results when tested with a PNPB esterase assay than less powerful enzymes like LC cutinase (joo et. al.). This assay has been very popular with iGEM teams, but can not be used to determine which of two enzymes is more effective at PET degradation.
+
First, the fastest PET degrading enzyme currently on the registry, PETase, is highly specific for PET. This means that despite its superior degrading ability, it will actually produce weaker results when tested with a PNPB esterase assay than less powerful enzymes like LC cutinase (joo et. al.). This assay has been very popular with iGEM teams, but can not be used to determine which of two enzymes is more effective at PET degradation. (Please use PNPB results only to verify PETase function, not to compare PETase efficiencies!)
 
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Second, enzymatic degradation of PET is glacially slow. No iGEM team has been able to confirm PET degradation on the basis of visual inspection or weight. The only way this degradation has been successfully detected is with powerful instruments such as SEM, mass spec, HPLC, and cell-free expression. Teams without access to these expensive resources have no way to obtain results yet.
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Second, enzymatic degradation of PET is glacially slow. No iGEM team has been able to confirm PET degradation on the basis of visual inspection or weight. The only way this degradation has been successfully detected is with powerful instruments such as SEM, mass spec, HPLC, and cell-free expression combined with UV vis. Teams without access to these expensive resources have no way to obtain results yet.
 
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<img src="https://static.igem.org/mediawiki/2018/e/e0/T--UMaryland--PET_MHET_product.png" style="height: inherit; width: inherit;" alt="Waluigi Time!">
 
<img src="https://static.igem.org/mediawiki/2018/e/e0/T--UMaryland--PET_MHET_product.png" style="height: inherit; width: inherit;" alt="Waluigi Time!">
 
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A popular figure on PETase byproducts and the role of MHETase</div>
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PETase byproducts and the role of MHETase [2]</div>
 
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The third challenge lies in the nature of PET degradation byproducts. These include MHET, ethylene Glycol, and TPA. Ethylene glycol is metabolized by E. coli, and teams have not been able to to determine enzymatic efficiency based on cell growth from ethylene glycol production. MHET is converted to TPA and ethylene glycol by MHETase, which makes TPA the most relevant target when quantifying degradation. However, TPA has poor solubility in water of about 100uM. Thus, any method to detect TPA from PET degradation must be extremely sensitive.
 
The third challenge lies in the nature of PET degradation byproducts. These include MHET, ethylene Glycol, and TPA. Ethylene glycol is metabolized by E. coli, and teams have not been able to to determine enzymatic efficiency based on cell growth from ethylene glycol production. MHET is converted to TPA and ethylene glycol by MHETase, which makes TPA the most relevant target when quantifying degradation. However, TPA has poor solubility in water of about 100uM. Thus, any method to detect TPA from PET degradation must be extremely sensitive.
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Our project employs a fluorescent biosensor from Los Alamos, PcaU, that is the product of directed evolution for high sensitivity to a downstream byproduct of TPA metabolism: Protocatechuic acid (PCA). We have shown that this sensor differentiates single micromolar PCA concentrations and can be used to detect TPA. Teams will be able to quantify PET degradation through bacterial fluorescence without need for expensive instruments. The quantitative cell-based sensor has potential use in the directed evolution of PETase as well. Notably, many of the latest articles of PETase mention that the enzyme holds promise for substantial improvement, making the promise of such an approach quite exciting. Details on the PcaU biosensor are available in its <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2825002"><u>registry page</u></a>.
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Our project employs a fluorescent biosensor from Jha et. al [3], PcaU, that is the product of directed evolution for high sensitivity to a downstream byproduct of TPA metabolism: Protocatechuic acid (PCA). We have shown that this sensor differentiates single micromolar PCA concentrations and can be used to detect TPA. Teams will be able to quantify PET degradation through bacterial fluorescence without need for expensive instruments.  
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Also, the fluorescent cell-based sensor has potential future use in the directed evolution of PETase via flow cytometry since cell fluorescence varies with concentration of PET degradation byproduct. This powerful method can sort up to 10^9 mutants a day! Notably, many of the latest articles of PETase mention that the enzyme holds promise for substantial improvement [2], making the prospect of such an approach quite exciting. Further details, characterization, and results on the PcaU biosensor have been added in its <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2825002"><u>registry page</u></a>.
 
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<img src="https://static.igem.org/mediawiki/2018/6/6d/T--UMaryland--PCAU_low.png" style="width: 80%" alt="Waluigi Time!">
 
<img src="https://static.igem.org/mediawiki/2018/6/6d/T--UMaryland--PCAU_low.png" style="width: 80%" alt="Waluigi Time!">
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<div class="imageBoxDescription">The lower boundary of PCAU detection range, n=8. Such sensitivity is necessary if we hope to detect and quantify TPA production from PET degradation.</div>
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<div class="imageBoxDescription">Our results determined the lower boundary of PcaU detection range is one micromolar TPA (n=8). Such sensitivity is necessary if we hope to detect and quantify TPA production from PET degradation.</div>
 
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Sources</div>
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1. Yoshida, S. et al. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 351, 1196–1199 (2016).
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2. Beckham, G. et al. Characterization and engineering of a plastic-degrading aromatic polyesterase. PNAS 115 (2018).
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3. Jha, R. K., Kern, T. L., Fox, D. T., & M. Strauss, C. E. (2014). Engineering an Acinetobacter regulon for biosensing and high-throughput enzyme screening in E. coli via flow cytometry. Nucleic Acids Research, 42(12), 8150–8160. http://doi.org/10.1093/nar/gku444
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Latest revision as of 02:12, 18 October 2018

Template Title Template Title

Measurement
Accessible, Sensitive Biosensor for PET Degredation
PET degradation has been and is a popular iGEM project. However, measurement of this degradation remains a challenge.
Current Problems
Waluigi Time!
Figure from Yoshida et. al. [1]
Despite being much more effective at degradation of PET, PETase produces minimal results in a PNPB absorbance assay.
Measurement of PET degradation is difficult for three major reasons.

First, the fastest PET degrading enzyme currently on the registry, PETase, is highly specific for PET. This means that despite its superior degrading ability, it will actually produce weaker results when tested with a PNPB esterase assay than less powerful enzymes like LC cutinase (joo et. al.). This assay has been very popular with iGEM teams, but can not be used to determine which of two enzymes is more effective at PET degradation. (Please use PNPB results only to verify PETase function, not to compare PETase efficiencies!)

Second, enzymatic degradation of PET is glacially slow. No iGEM team has been able to confirm PET degradation on the basis of visual inspection or weight. The only way this degradation has been successfully detected is with powerful instruments such as SEM, mass spec, HPLC, and cell-free expression combined with UV vis. Teams without access to these expensive resources have no way to obtain results yet.

Waluigi Time!
Very pricey white benchtop machines that detect PET degradation (SEM, LCMS, HPLC). Very, very pricey.
Waluigi Time!
PETase byproducts and the role of MHETase [2]
The third challenge lies in the nature of PET degradation byproducts. These include MHET, ethylene Glycol, and TPA. Ethylene glycol is metabolized by E. coli, and teams have not been able to to determine enzymatic efficiency based on cell growth from ethylene glycol production. MHET is converted to TPA and ethylene glycol by MHETase, which makes TPA the most relevant target when quantifying degradation. However, TPA has poor solubility in water of about 100uM. Thus, any method to detect TPA from PET degradation must be extremely sensitive.
Our Solution
Our project employs a fluorescent biosensor from Jha et. al [3], PcaU, that is the product of directed evolution for high sensitivity to a downstream byproduct of TPA metabolism: Protocatechuic acid (PCA). We have shown that this sensor differentiates single micromolar PCA concentrations and can be used to detect TPA. Teams will be able to quantify PET degradation through bacterial fluorescence without need for expensive instruments.
Also, the fluorescent cell-based sensor has potential future use in the directed evolution of PETase via flow cytometry since cell fluorescence varies with concentration of PET degradation byproduct. This powerful method can sort up to 10^9 mutants a day! Notably, many of the latest articles of PETase mention that the enzyme holds promise for substantial improvement [2], making the prospect of such an approach quite exciting. Further details, characterization, and results on the PcaU biosensor have been added in its registry page.

Waluigi Time!
Our results determined the lower boundary of PcaU detection range is one micromolar TPA (n=8). Such sensitivity is necessary if we hope to detect and quantify TPA production from PET degradation.
Sources
1. Yoshida, S. et al. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 351, 1196–1199 (2016).
2. Beckham, G. et al. Characterization and engineering of a plastic-degrading aromatic polyesterase. PNAS 115 (2018).
3. Jha, R. K., Kern, T. L., Fox, D. T., & M. Strauss, C. E. (2014). Engineering an Acinetobacter regulon for biosensing and high-throughput enzyme screening in E. coli via flow cytometry. Nucleic Acids Research, 42(12), 8150–8160. http://doi.org/10.1093/nar/gku444

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