Difference between revisions of "Team:UMaryland/TPA"

 
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12 well plate TPA activity assay, n=4. Each well in a 12 well plate was filled with 900ul of 10mM Tris, pH 7.2, with 100uM TPA. 100um of enzyme mix supernatant was added to 4 wells, 100um of enzyme mix resuspended pellet was added to another 4, and water was added to 4 control wells. Plate was incubated at 30C overnight to enzymatically convert TPA to PCA. PcaU BL21 cells were grown to OD600=0.6, and 1mL culture was added to each well, diluting original TPA concentration to a final 45uM. Fluorescence was taken in a plate reader eight hours later at 395nm excitation, 509nm emission. A significant difference was observed between supernatant and control. The pellet failed to produce a significant result.
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The plasmids were expressed in BL21 DE3 E. coli and lysate was prepared using the protocol below.
 
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<img src="https://static.igem.org/mediawiki/2018/9/9a/T--UMaryland--TPA_detection.png" style="max-width: 80%" alt="Waluigi Time!">
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<img src="https://static.igem.org/mediawiki/2018/d/d0/T--UMaryland--tphspin.png" style="max-width: 80%" alt="Waluigi Time!">
 
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Expression was verified using the same SDS-PAGE protocol employed by Darmstadt 2012, for which we obtained similar results.
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TPA detection assay, n=4. Each well was filled with 900ul of 10mM Tris, pH 7.2, with 4 containing 100um TPA, 4 containing 50um TPA, and 4 containing no TPA. 100ul of tph enzyme mix was added to each well and plate was incubated at 30C overnight to enzymatically convert TPA to PCA. PcaU BL21 cells were grown to OD600=0.6, and 1mL culture was added to each well, diluting samples to 45uM TPA and 22.5uM TPA final concentrations. Fluorescence was taken in a plate reader six hours later at 395nm excitation, 509nm emission. A significant difference was observed between culture exposed to degraded TPA and the control. The difference between 22.5uM TPA and 45uM TPA was not significant.
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This protocol has demonstrated efficacy for detecting the presence of a PET degradation byproduct, TPA. However, the magnitude of error results in an inability to distinguish between certain concentrations of product. If the sensor is to be used for directed evolution, it must be capable of doing this. Improving sensor resolution to achieve this result could be accomplished in a variety of manners. The first action we would pursue is HIS purification of the TPH enzymes, since earlier tests suggested that the presence of cell lysate in the TPH enzyme mix may have impacted the growth of E. coli and driven up error. A lab with more resources and time would be able to express TPH enzymes and PCAU in the same cell. Not only would this drive down error from exposure to cell lysate, but it would permit single cell analysis of TPH activity through flow cytometry.
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TPH SDS Gels
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SDS gels of TPH enzymes in E. coli whole cell fraction from the protocol shown earlier. Lanes are denoted by letter code and hours of incubation post induction. For example, B4h = TphB 4 hours post induction. Blue boxes denote bands at molecular weight matching the Darmstad's result. The purple box is an unexpected, unknown band. Severe leaky expression of TphA1A2 and TphB can be seen in the zero hour fractions. However, our results in the demonstration section show that this has not affected the enzyme's function.</div>
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<img src="https://static.igem.org/mediawiki/2018/2/24/T--UMaryland--PETasePCAU2.png" style="max-width: 80%" alt="Waluigi Time!">
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PETase activity test, testing supernatant of PET plastic exposed to PETase lysate for one week. Error level is high due to the 24 well plate hitting the wall of the shaker, resulting in the plate needing to be read 2 hours after PcaU induction. The hit caused some media to leak from the sides of wells, which was partially corrected for by dividing fluorescence over absorbance. The results on this test are not conclusive, but they are promising. Knowing that 25uM TPA is distinguishable from a negative control after 6 hours with our approach, the fact that the average fluorescence for degraded PET is noticeably higher than our TPA positive control leads us to predict that the assay is capable of producing results.
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We tested TPH enzyme functionality in various buffers to develop a final buffer, TPH buffer. TPH buffer is a buffer that must maintain TPH enzyme function in the presence of PETase buffer. This is because the resuspended TPH enzymes will be applied to supernatant from PETase degradation containing TPA. The TPH enzymes will convert TPA to PCA for detection by PcaU. Therefore, these buffers must also not prevent growth of PcaU transformed E. coli cells.
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10mM Tris buffer at pH 7.2 was chosen for PETase buffer and TPH buffer. PNPB tests from multiple teams including our own have shown that PETase is functional in Tris, and our own growth tests revealed that although Tris may be somewhat toxic, PcaU transformed BL21 E. coli are able to grow in the presence of 10mM tris buffer. TPH buffer also contains some key components used in Masai’s work, including 0.1mM ferrous ammonium sulfate (required iron supply), 2mM L-Cysteine Hydrochloride, and 10% glycerol (for preservation). We also added 100um NADPH to this buffer to speed the reaction
 
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It is unusual that PETase would produce such high fluorescence, however, because that would require a great amount more TPA that 25uM. In fact, it would require more than should be able to dissolve in water. This may have been a result of active PETase still being present in the supernatant when the TPH enzyme mix was added. Thus, as the TPH enzyme mix was converting TPA to PCA during the overnight incubation, PETase may have been producing more TPA from PET byproducts left over in the supernatant.
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PETase has worked in PBS, but TPH enzymes may not due to NADPH being unstable in phosphate buffers. PBS also precipitated when ferrous ammonium sulfate was added to it, likely due to formation of insoluble ferrous ammonium phosphate. PBS is not a suitable buffer for TPH enzymes
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The test above will be repeated, but data will not be available in time for the wiki freeze. We are optimistic that we can conclusively detect PET degradation, and if we manage to do so you will see our results at the Jamboree! In conclusion, there are many, many variables to account for in this system. However, with enough continued testing and optimization we are confident it will be possible to quantify PET degradation using this system. Then, the potential of directed PETase evolution via cell fluorescence will be unleashed!
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1. Sasoh, M., Masai, E., Ishibashi, S., Hara, H., Kamimura, N., Miyauchi, K., & Fukuda, M. (2006). Characterization of the Terephthalate Degradation Genes of Comamonas sp. Strain E6. Applied and Environmental Microbiology, 72(3), 1825–1832.
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2. Schläfli, H. R., Weiss, M. A., Leisinger, T., & Cook, A. M. (1994). Terephthalate 1,2-dioxygenase system from Comamonas testosteroni T-2: purification and some properties of the oxygenase component. Journal of Bacteriology, 176(21), 6644–6652.
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3. Yuki Fukuhara, Daisuke Kasai, Yoshihiro Katayama, Masao Fukuda & Eiji Masai (2008) Enzymatic Properties of Terephthalate 1,2-Dioxygenase of Comamonas sp. Strain E6, Bioscience, Biotechnology, and Biochemistry, 72:9,2335-2341, DOI: 10.1271/bbb.80236
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Latest revision as of 03:07, 18 October 2018

Template Title Template Title

TPA ENZYME MIX
Enzymatically converting TPA to PCA for detection
The enzymes needed to convert TPA to PCA are available in the registry from Darmstadt 2012. By the time PcaU was syntesized, we did not have enough time to use Darmstadt’s parts which required assembly. We contacted professor Eiji Masai, who generously provided us with the necessary plasmids to express the enzymes in E. coli. These are pETtpa23, pRSFtpa1 and pETtpb from Masai’s 2006 paper.

Waluigi Time!
Figure from Masai et. al.
The terepthalic acid degradation pathway. It is an aerobic process that requires the presence of NADPH, although the NADPH is restored at the end of the proccess.

The plasmids were expressed in BL21 DE3 E. coli and lysate was prepared using the protocol below.

Waluigi Time!
Expression was verified using the same SDS-PAGE protocol employed by Darmstadt 2012, for which we obtained similar results.
Waluigi Time!
TPH SDS Gels
SDS gels of TPH enzymes in E. coli whole cell fraction from the protocol shown earlier. Lanes are denoted by letter code and hours of incubation post induction. For example, B4h = TphB 4 hours post induction. Blue boxes denote bands at molecular weight matching the Darmstad's result. The purple box is an unexpected, unknown band. Severe leaky expression of TphA1A2 and TphB can be seen in the zero hour fractions. However, our results in the demonstration section show that this has not affected the enzyme's function.

We tested TPH enzyme functionality in various buffers to develop a final buffer, TPH buffer. TPH buffer is a buffer that must maintain TPH enzyme function in the presence of PETase buffer. This is because the resuspended TPH enzymes will be applied to supernatant from PETase degradation containing TPA. The TPH enzymes will convert TPA to PCA for detection by PcaU. Therefore, these buffers must also not prevent growth of PcaU transformed E. coli cells.
10mM Tris buffer at pH 7.2 was chosen for PETase buffer and TPH buffer. PNPB tests from multiple teams including our own have shown that PETase is functional in Tris, and our own growth tests revealed that although Tris may be somewhat toxic, PcaU transformed BL21 E. coli are able to grow in the presence of 10mM tris buffer. TPH buffer also contains some key components used in Masai’s work, including 0.1mM ferrous ammonium sulfate (required iron supply), 2mM L-Cysteine Hydrochloride, and 10% glycerol (for preservation). We also added 100um NADPH to this buffer to speed the reaction

PETase has worked in PBS, but TPH enzymes may not due to NADPH being unstable in phosphate buffers. PBS also precipitated when ferrous ammonium sulfate was added to it, likely due to formation of insoluble ferrous ammonium phosphate. PBS is not a suitable buffer for TPH enzymes
sources
1. Sasoh, M., Masai, E., Ishibashi, S., Hara, H., Kamimura, N., Miyauchi, K., & Fukuda, M. (2006). Characterization of the Terephthalate Degradation Genes of Comamonas sp. Strain E6. Applied and Environmental Microbiology, 72(3), 1825–1832.
2. Schläfli, H. R., Weiss, M. A., Leisinger, T., & Cook, A. M. (1994). Terephthalate 1,2-dioxygenase system from Comamonas testosteroni T-2: purification and some properties of the oxygenase component. Journal of Bacteriology, 176(21), 6644–6652.
3. Yuki Fukuhara, Daisuke Kasai, Yoshihiro Katayama, Masao Fukuda & Eiji Masai (2008) Enzymatic Properties of Terephthalate 1,2-Dioxygenase of Comamonas sp. Strain E6, Bioscience, Biotechnology, and Biochemistry, 72:9,2335-2341, DOI: 10.1271/bbb.80236

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