Difference between revisions of "Part:BBa M50056:Experience"

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===Applications of BBa_M50056===
 
===Applications of BBa_M50056===
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The unlysed double mutant cells show some catalytic activity based on the plate reader result of pNPB assay
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    According to Yoshida et al., PETase degrades PET into MHET, yet we anticipated challenges measuring PETase activity by MHET absorbance. Based on Tianjin’s documentation, we knew that PET degradation was slow. In addition, NIST representatives pointed out to us that we might have difficulty quantifying absorbance at 260nm, as DNA and protein also absorb at this wavelength. (Team Tianjin ran their experiment in a cell-free system; they therefore did not experience the same problem.) For these reasons, we ordered not only PET products like PET film, but also 4-Nitrophenyl butyrate (pNPB), the substrate iGEM Team Harvard used as a stand-in for PET in their analogous plate reader experiment. pNPB contains an ester bond resembling that found in PET, the bond broken by PETase via hydrolysis. We planned to use pNPB hydrolysis as a model for PET degradation.  pNPB hydrolysis is easier to observe than PET hydrolysis, as the product absorbs a more clearly distinguishable wavelength at 400/405nm.
  
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[[File:12 08 double unlysed.tiff]]
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      We followed the UC Davis iGEM Team’s pNPB assay protocol, which was the protocol used by Team Harvard in 2016. We did the pNPB assay for both unlysed cells and cell lysates to bypass concern surrounding our ompT secretion tag. Here's our pNPB assay plot of the double mutant based on the plate reader reading result:
  
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[[File:12 08 double lysate.jpeg]]
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            [[File:12 08 double unlysed.tiff]]
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      The above figure shows the absorbance result for unlysed double mutant cells. For both induced and uninduced samples of unlysed cells (left graph), we see increases in absorption only in the presence of pNPB, as we saw in previous assays. This increasing absorption suggests degradation of pNPB. When pNPB is substituted for LB in our control assays, absorption remains constant, as expected. Encouragingly, on average, the wells with induced cells + pNPB show higher absorption than wells with non-induced cells + pNPB.
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      The lysis buffer introduced variability in the OD values, so we assembled data for lysed and non-lysed cells separately. Unfortunately, our lysed cell data proved messy and unreliable:
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            [[File:12 08 double lysate.jpeg]]
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      For the double mutant cell lysates, induced lysate with no substrate produced highest absorbance values. No clear trends or patterns are obvious from these plots, and we hypothesize that background noise from other proteins present in lysate could obscure absorbance readings at 405 nm. In the future, we would repeat pNPB assays using purified protein only.
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===References===
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===User Reviews===
 
===User Reviews===

Revision as of 06:24, 12 December 2016


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Please enter how you used this part and how it worked out.

Applications of BBa_M50056

    According to Yoshida et al., PETase degrades PET into MHET, yet we anticipated challenges measuring PETase activity by MHET absorbance. Based on Tianjin’s documentation, we knew that PET degradation was slow. In addition, NIST representatives pointed out to us that we might have difficulty quantifying absorbance at 260nm, as DNA and protein also absorb at this wavelength. (Team Tianjin ran their experiment in a cell-free system; they therefore did not experience the same problem.) For these reasons, we ordered not only PET products like PET film, but also 4-Nitrophenyl butyrate (pNPB), the substrate iGEM Team Harvard used as a stand-in for PET in their analogous plate reader experiment. pNPB contains an ester bond resembling that found in PET, the bond broken by PETase via hydrolysis. We planned to use pNPB hydrolysis as a model for PET degradation.  pNPB hydrolysis is easier to observe than PET hydrolysis, as the product absorbs a more clearly distinguishable wavelength at 400/405nm. 

     We followed the UC Davis iGEM Team’s pNPB assay protocol, which was the protocol used by Team Harvard in 2016. We did the pNPB assay for both unlysed cells and cell lysates to bypass concern surrounding our ompT secretion tag. Here's our pNPB assay plot of the double mutant based on the plate reader reading result:

            12 08 double unlysed.tiff

     The above figure shows the absorbance result for unlysed double mutant cells. For both induced and uninduced samples of unlysed cells (left graph), we see increases in absorption only in the presence of pNPB, as we saw in previous assays. This increasing absorption suggests degradation of pNPB. When pNPB is substituted for LB in our control assays, absorption remains constant, as expected. Encouragingly, on average, the wells with induced cells + pNPB show higher absorption than wells with non-induced cells + pNPB. 

     The lysis buffer introduced variability in the OD values, so we assembled data for lysed and non-lysed cells separately. Unfortunately, our lysed cell data proved messy and unreliable: 

            12 08 double lysate.jpeg

     For the double mutant cell lysates, induced lysate with no substrate produced highest absorbance values. No clear trends or patterns are obvious from these plots, and we hypothesize that background noise from other proteins present in lysate could obscure absorbance readings at 405 nm. In the future, we would repeat pNPB assays using purified protein only.


References

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User Reviews

UNIQ1ef8572db33962c7-partinfo-00000000-QINU UNIQ1ef8572db33962c7-partinfo-00000001-QINU