Difference between revisions of "Part:BBa K4949004"
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Est119 is an esterase originally identified in the Thermobifida alba strain AHK119. Est119 has been shown to degrade aliphatic-aromatic copolyesters and decrease the size of polymer particles of other biodegradable plastics, with an optimal temperature range of 45-55°C
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Est119 is an esterase originally identified in the <i>Thermobifida alba</i> strain AHK119 (AB298783). Est119 has been shown to degrade aliphatic-aromatic copolyesters and decrease the size of polymer particles of other biodegradable plastics, with an optimal temperature range of 45-55°C (Hu and al. 2009). Similarly to MGS0156, Est119 is interesting due to its potential temperature compatibility with Manitoban composting methods. Our team modified the Est119 genetic sequence to include the Lpp-OmpA anchor to allow for the characterization of the surface-display mechanism of PLAnet Zero
  
 
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<html><img src="https://static.igem.wiki/teams/4949/wiki/registry/ttl-pnob-mmeaa-graphs.png" width="950" height="700"></html>
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'''Figure 1.''' Michaelis-Menten Esterase Activity Assay of <i>Thermoanaerobacter thermohydrosulfuricus</i> lipase with 4-nitrophenyl butyrate (pNOB). Assays were conducted with 1.5 µM of lipase in PBS with pNOB concentration ranging from 0.0365-0.11 µM. The rate of change of absorbance was monitored at 410 nm for 1 min. A) Wild-type TTL lipase chromogenic assay with initial rate as a function of substrate concentration at 25 B) Wild-type TTL lipase chromogenic assay with initial rate as a function of substrate concentration at 60 ℃ C) TTL lipase chromogenic assay with initial rate as a function of substrate concentration, with norleucine introduced by residue specifically by SPI at 25 ℃ D) TTL lipase chromogenic assay with initial rate as a function of substrate concentration, with norleucine introduced by residue.
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<html><img src="https://static.igem.wiki/teams/4949/wiki/results/figure-18-est119-wholecell-lpp-delta-strain.png" width="950" height="700"></html>
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'''Figure 1.''' Absorbance as a function of time for the whole cell esterase activity assay of Est119. After induced expression, <i>E.coli</i> BL21 (DE3) + Est 119 or <i>E.coli</i> BL21 (DE3)ΔLpp + Est119,  a native Lpp deficient stain, were washed and resuspended in PBS with 100µM NPO with a cell density normalized to OD<sub>600</sub>=1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant as measured at 410 nm.
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<html><img src="https://static.igem.wiki/teams/4949/wiki/results/figure19-est119-npo-deltalpp-plasmid.png" width="950" height="700"></html>
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'''Figure 2.''' Absorbance as a function of time for the whole cell esterase activity assay of Est119. After induced expression, <i>E.coli</i> BL21 (DE3) + Est119 or <i>E.coli</i> BL21 (DE3)ΔLpp +Est119, a native Lpp deficient stain, were washed and resuspended in PBS with 100µM pNOB with a cell density normalized to OD<sub>600</sub>=1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant as measured at 410 nm.  
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<html><img src="https://static.igem.wiki/teams/4949/wiki/results/figure20-est119-npo-deltalpp-plasmid.png" width="950" height="700"></html>
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'''Figure 20.''' Absorbance as a function of time for the whole cell esterase activity assay of Est119. After induced expression <i>E.coli</i> BL21 (DE3) + Est119 or <i>E.coli</i> BL21 (DE3) + Est119(ΔLpp), bacteria with a Lpp deficient plasmid, were washed, and resuspended in PBS with 100µM pNOB with a cell density normalized to OD<sub>600</sub>=1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant as measured at 410 nm.
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<html><img src="https://static.igem.wiki/teams/4949/wiki/results/figure21-est119-npo-deltalpp-plasmid.png" width="950" height="700"></html>
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'''Figure 4.''' Absorbance as a function of time for the whole cell esterase activity assay of Est119. After induced expression, <i>E.coli</i> BL21 (DE3) + Est119 or <i>E.coli</i> BL21 (DE3) + Est119(ΔLpp), bacteria with an Lpp deficient plasmid,  were washed and resuspended in PBS with 100µM NPO with a cell density normalized to OD<sub>600</sub>=1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant as measured at 410 nm.
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Revision as of 03:00, 12 October 2023

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Est119 is an esterase originally identified in the Thermobifida alba strain AHK119 (AB298783). Est119 has been shown to degrade aliphatic-aromatic copolyesters and decrease the size of polymer particles of other biodegradable plastics, with an optimal temperature range of 45-55°C (Hu and al. 2009). Similarly to MGS0156, Est119 is interesting due to its potential temperature compatibility with Manitoban composting methods. Our team modified the Est119 genetic sequence to include the Lpp-OmpA anchor to allow for the characterization of the surface-display mechanism of PLAnet Zero

Sequence and Features Status: 500 Content-type: text/html

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Figure 1. Absorbance as a function of time for the whole cell esterase activity assay of Est119. After induced expression, E.coli BL21 (DE3) + Est 119 or E.coli BL21 (DE3)ΔLpp + Est119, a native Lpp deficient stain, were washed and resuspended in PBS with 100µM NPO with a cell density normalized to OD600=1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant as measured at 410 nm.

Figure 2. Absorbance as a function of time for the whole cell esterase activity assay of Est119. After induced expression, E.coli BL21 (DE3) + Est119 or E.coli BL21 (DE3)ΔLpp +Est119, a native Lpp deficient stain, were washed and resuspended in PBS with 100µM pNOB with a cell density normalized to OD600=1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant as measured at 410 nm.

Figure 20. Absorbance as a function of time for the whole cell esterase activity assay of Est119. After induced expression E.coli BL21 (DE3) + Est119 or E.coli BL21 (DE3) + Est119(ΔLpp), bacteria with a Lpp deficient plasmid, were washed, and resuspended in PBS with 100µM pNOB with a cell density normalized to OD600=1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant as measured at 410 nm.

Figure 4. Absorbance as a function of time for the whole cell esterase activity assay of Est119. After induced expression, E.coli BL21 (DE3) + Est119 or E.coli BL21 (DE3) + Est119(ΔLpp), bacteria with an Lpp deficient plasmid, were washed and resuspended in PBS with 100µM NPO with a cell density normalized to OD600=1.0. Samples were then incubated for 5 min at room temperature. Aliquots of 100 µl were collected at 5 intervals and the absorbance of the supernatant as measured at 410 nm.