Difference between revisions of "Part:BBa K3576001"

Revision as of 13:23, 4 September 2022

PETase expression system

It is the key part that is responsible for expressing PETase. The PETase can hydrolyze PET to MHET. In order to ensure that PETase can be fully in contact with extracellular PET. The sequence of coding pelB signal peptide was added before PETase.

Sequence and Features

Results

1. Protein expression test

SDS-PAGE electrophoresis was used to check the expression of PETase protein. As shown in Figure 1, compared to the blank control, the lane contained PETase (40 KDa) protein indicated that this protein has been successfully expressed.

Figure 1 Protein SDS-PAGE electrophoresis results of PETase.

2. Enzyme Activity Test of PETase

The enzyme activity of PETase was performed by p-NP assay which is a common way to quantify hydrolytic activity. We selected p-Nitrophenylbutyrate (pNPB) as the substrate, which can be hydrolyzed to p-nitrophenol (pNP) (Figure 2-A). The concentration of pNP can be measured by the characteristic absorption at 405 nm. As shown in Figure 2-B, with the extension of reaction time, the OD405 value of p-NP gradually increased, which indicates that the degradation activity of the PETase.

Figure 2 (A)The mechanism of pNPB degradation; (B) OD405 of pNPB hydrolysis by overexpressed PETase.

2022 Bioplus-China’s Characterization of BBa_ K3576001

Introduction

This program designed a method for degradation of Polyethylene Terephthalate (PET) by an engineered bacteria which expresses PETase encoded in constructed plasmid transformed into the Escherichia coli. Whether engineered E. coli express PEATase as designed, is assessed by repeating the experimental process and comparisons of the catalytic ability.

Method

The purpose of our experiment is to produce engineered bacteria to express PET enzyme, so that the expressed PETase can catalyze the degradation of PET. The competent cells of DH5a and BL21 were first prepared in the main part of the experiment. The competent bacteria were further transformed with constructed plasmids containing resistance gene to ampicillin and coding gene for PETase. The transformed bacteria then expanded further. In order to produce adequate enzymes, we chosed the BL21 strain for PETase production, an engineering bacterium that were optimized for protein expression. In the whole experiment process, we validated all experimental procedures by a series of standard operations. To verify successful transformation of plasmid DNA, we used DH5a strain to do two related tests. The colony PCR and plasmid digestion experiments were carried out respectively. The products of PCR and digestion were then verified using DNA electrophoresis. For PETase production, we selected BL21 strain which were transformed with expression plasimid successfully. At last, SDS-PAGE verification was performed after lysis of the expressed bacteria and the untransformed negative control bacteria.

Results

The transformed E. coli were subjected to plasmid extraction and agarose gel electrophoresis, and the bands marked in the figure appeared between 1000-1500 bp, indicating that E. coli from all six sample sources were probably successfully transformed. The PCR product size was considered as 1493bp as show in Figure1.

Figure 1 result of colony PCR Line1: DNA marker 1; Line2 and 9: DNA marker 2; Line3, 4, 5, 6, 7 and 8: plasmid extracted from transformed Escherichia coli

The extracted plasmid was electrophoresed after enzymatic digestion, and the size of the band labeled in the figure (1000-1500bp) is consistent with the nucleic acid sequence between the two restriction endonuclease specific binding sites of EcoR1 and BamH1(1249bp) and the absence of this band in the negative control, indicating that the nucleic acid sequence expressing PETase was probably successfully inserted in the plasmid. The Enzyme section size was considered as 1249 bp as show in Figure 2.

Figure result of enzyme digestion Line 1 and 12: 1kb plus DNA marker; Line2: D2000 DNA marker; Line 3, 4, 5, 6, 7, 8, 9 and 10: plasmid samples after enzyme digestion; Line11: negative control group

The protein extracted from the successfully transformed E. coli treated with Ripa lysate and PMSF showed bands between 40 and 55 kDa in size （which is the PETase band） after SDS-Page protein electrophoresis, and there was no such band in the negative control group, which was consistent with the expected results, indicating that E. coli successfully expressed PETase as show in Figure 3 .

Figure 3 result of SDS-PAGE testing Line1&2: Page-Ruler Prestained Protein Ladder; Line3&4: negative control; Line5&6: Protein extracted by Ripa lysate and PMSF

After three experiments to take the average value, the absorbance curve of the experimental group at 405nm wavelength showed an overall rising trend, with a fast growth rate in 0-15min, and leveled off to reach a plateau at about 45min; the curve of the control group was flat without fluctuation and at a lower value as show in Figure 4. Blue line/red line: experimental groups containing PETase-expressing bacterial broth, acetonitrile and 4-nitrophenyl butyrate to explore the enzymatic activity of PETase at different concentrations.Gray line: control group containing bacterial solution and acetonitrile to exclude the effect of absorbance of 4-nitrophenyl butyrate on the experimental results. Yellow line: control group containing saline, acetonitrile and 4-nitrophenyl butyrate to exclude the effect of absorbance of the bacterial solution on the experimental results

Figure 4 result of pNp catalytical reaction

Conclusion

We introduced the plasmid DNA expressing PETase into E. coli, and the presence of the target sequence was verified by PCR and following agarose gel electrophoresis. We further confirmed the success of the transformation after two experiments, enzyme digestion electrophoresis of the extracted plasmid and SDS-Page electrophoresis of the protein extracted from the successfully transformed bacterial broth. After that, we used p-NP assay to verify the enzyme activity by measuring the absorbance value of p-nitrophenol, the colored product of p-Nitrophenylbutyrate hydrolyzed by PETase, at 405 nm. After the experiments results were analyzed. The dynamic curve of the experimental group showed an overall increasing trend and was positively correlated with the concentration of the bacterial solution, reaching a plateau at about 45 min, while these performances did not appear in the control group. This means that the E. coli from the experimental group successfully expressed the PETase with hydrolytic activity.