Latest revision as of 11:16, 20 October 2021

T7 pro-tag-Lac ope-PPO-T7 ter

BBa_K3584002 is used to express Polyphenol oxidases(PPD) protein for Ni-sepharose purification.

Sequence and Features

Contribution

BBa_K3584000 is the coding sequence of a polyphenol oxidase (PPO) from Pleurotus ostreatus, which is different from the present enzymes showing the similar activity. PPO is a group of enzyme that are able to oxidize phenol or polyphenol by molecular oxygen to form the corresponding quinone. In a broad sense, polyphenol oxidase can be divided into three categories: monophenol monooxidase (tyrosinase, EC.1.14.18.1), bisphenol oxidase (catechol oxidase, EC. 1.10.3.2) and laccase (laccase, EC.1.10.3.1). Among these three types of polyphenol oxidase, catechol oxidase is mainly distributed in plants, and the polyphenol oxidase in microorganisms mainly includes laccase and tyrosinase. The polyphenol oxidase mentioned in most of the literature is generally the collective name of catechol oxidase and laccase.

Figure 0

Engineering Success

Production, purification, and SDS-PAGE analysis of recombinant PPO In order to present the function of the part, the PPO gene was expressed in E. coli BL21 (DE3) under the control of T7 promoter. Then the bacterial cells are collected and crushed, and the PPO enzyme solution is purified by further confirmation by the SDS-PAGE method, which is found in the corresponding protein band of approximately 57 kDa (Figure 1).

Figure 1

Figure 1. Production, purification, and SDS-PAGE analysis of recombinant PPO. Lane: protein molecular weight standard; lane 8: purified PPO.

Enzymatic activity of the PPO PPO activity was determined using an analogue of p-cresol (catechol) as a substrate. In proper reaction mixture, PPO and 10 mM catechol was incubated at 50 ℃, pH 7.0 for 3 min, and the change in absorbance at 420 nm was measured spectrophotometrically. One unit of enzyme (PPO) activity was defined as the amount of enzyme that increased absorbance of 0.001 per minute. We found that as the extension of reaction time, reaction liquid of absorbance at 420 nm increased linear (Figure 2), we obtained by catalytic activity of PPO enzyme fluid, and divided by the corresponding protein concentration, the specific activity of PPO (Table 1).

Figure 2

Figure 2. PPO activity was determined using catechol as a substrate. The reaction mixture contained PPO protein and 10 mM catechol which was incubated at 50 ℃, pH 7.0 for 3 min, and the change in absorbance at 420 nm was recorded.

Table 1. Specific activity of recombinant PPO

Table 1

Characterization by 2021iGEM_Shanghai_Metro_HS

Improvement of an existing part

Compared to the old part BBa_K3584002, expressing T7 pro-tag-Lac ope-PPO-T7 ter, we design a new part BBa_K4096004, expressing T7 pro-Lac opePKC-OP-tag-T7 ter, with its sequence different from the old part (Figure 9). The group iGEM20_Shanghai_United aimed to degrade p-cresol sulfate for giving a potential treatment to chronic kidney disease patients. And they successfully constructed a composite part BBa_K3584002 and transformed it into E.coli BL21(DE3). They successfully expressed and purified PPO protein, and detected the enzyme activity. Differently, our team designed a new part BBa_K4096004 and aimed to construct an E.coli strain that can expressed alkali cellulase so as to improve ruminants' ability to acquire monosaccharides.. We chose T7 pro-Lac ope-PKC-OP-tag-T7 ter., which sequence is different from part BBa_K3584002 (Fig8), to construct our desired engineering strain.

Figure 9 The blast results about the sequence of our new part BBa_K4096004 and the old one BBa_K3584002..

Furthermore, in order to improve the utilization rate of silage and grain mixed feed, and the applicable varieties of poultry and ruminants. We constructed an improved composite part BBa_K4096006, which further add the Bacillus subtilis xylanase xynA gene to the cloning recombinant vector to make it compatible with cellulase PKC-OP and express β-xylosidase at the same time.

Profile

Name: pET-25b-PKC-OP

Base Pairs: 6633 bp

Origin: Synthetic

Properties: A recombinant plasmid containing cellulase sequence.

Usage and Biology

BBa_K4096004 is a plasmid that can expressed cellulase (PKC-OP) under the control of T7 promoter from Pseudomonas aeruginosa. Cellulases break down the cellulose molecule into monosaccharides ("simple sugars") such as beta-glucose, or shorter polysaccharides and oligosaccharides. Several different kinds of cellulases are known, which differ structurally and mechanistically. Synonyms, derivatives, and specific enzymes associated with the name "cellulase" include endo-1,4-beta-D-glucanase (beta-1,4-glucanase, beta-1,4-endoglucan hydrolase, endoglucanase D, 1,4-(1,3,1,4)-beta-D-glucan 4-glucanohydrolase), carboxymethyl cellulase (CMCase), avicelase, celludextrinase, cellulase A, cellulosin AP, alkali cellulase, cellulase A 3, 9.5 cellulase, and pancellase SS.

Construct design

The alkaline cellulase gene from Pseudomonas aeruginosa PKC-OP performed codon optimization on PKC-001 was selected and inserted into the pET-25b vector which contains a pelB signal peptide (Figure 1 and 2). The recombinant plasmid then was transformed it to E. coli BL21(DE3) strain to produce cellulase.

Figure 1. Genetic design of our gene.

Figure 2. Schematic map of pET-25b-PKC expression plasmid..

The profiles of every basic part are as follows:

BBa_K4096002

Name: PKC-OP

Base Pairs: 1086bp

Origin: Pseudomonas aeruginosa, genome

Properties: A coding sequence of alkali cellulase.

Usage and Biology

BBa_K4096002 is a coding sequence of alkali cellulase (PKC-OP) from Pseudomonas aeruginosa. Cellulases break down the cellulose molecule into monosaccharides ("simple sugars") such as beta-glucose, or shorter polysaccharides and oligosaccharides. Several different kinds of cellulases are known, which differ structurally and mechanistically.

BBa_K4096000

Name: pET-25b-vector

Base Pairs: 5547bp

Origin: Addgene

Properties: A plasmid expressing proteins.

Usage and Biology

BBa_K4096000 is a plasmid that can express proteins.

Experimental approach

Construction of recombinant plasmid

Figure 3. The result of enzyme cutting and PCR.

This picture is the nucleic acid electrophoresis result of enzyme cutting and PCR (By performing PCR, we can obtain the desirable PKC-OP’s gene segment.). Column “marker” is a column that is used to show the position of different lengths of genes. In this step, our aim is to verify whether these results are the desired ones. Number 1 and 2 is the result for pET-25b after enzyme digestion. Additionally, number 3 is the pET-25b without enzyme cutting. At last, number 4 and 5 is the PKC-OP after PCR. As our experiment moves on, the concentration of our PKC-OP and pET-25b are 233.2ng/μL and 17.8ng/μL. Lastly, we use the homologous combination to combine them.

Figure 4 E. coil having the desired pET-25b and PKC-OP.

The pET25b-PKC-OP was constructed. The plate shows monoclonals of pET25b-PKC-OP constructs.

Figure 5. The result of Apa1 enzyme digestion validation.

Column “marker” is a column that is used to show the position of different lengths of genes. Number 1 to 10 is the result for recombinant plasmid pET25b-PKC-OP after Apa1 enzyme digestion. We get two bands of 4712bp and 1894bp. It further indicates that the obtained monoclonals were positive monoclonals containing the recombinant plasmid. 1, 7 and 8 plasmids were sent to sequence.

Figure 6 The result of sequencing for plasmid pET25b-PKC-OP..

Sequencing feedback shows we have obtained the correct plasmids which is consistent with their DNA profiles. Different induction conditions were tested for protein expression. As the pET-25b vector contains a pelB signal peptide, the engineered strain would secret the protein into the medium. Therefore, we collected the culture supernatant after induction and ran SDS-PAGE for verification (Figure 7).

Figure 7. SDS-PAGE analysis of culture supernatant under different induction conditions..

The theoretical molecular weight of the PKC cellulase is 45.6 kDa. As seen from the SDS-PAGE (Figure 7), there is a wide band just above 40 kDa and it indicates that we have obtained the PKC cellulase.

Proof of function

As our goal is to produce the additive that could secret PKC enzyme to help degrade the cellulose in silage, we want to ensure that our engineered strain can make the best of itself at its best condition. From this perspective, we designed several different induction conditions for protein expression and decided to build the model to determine the optimal condition for our engineered strain to “work” better.

Table 1. The gray value of E. coli/PKC-OP under different concentrations of IPTG against time.

In table 1, gray values were measured by Image J in order to quantify the protein expression level. In this case, we tested three concentrations of IPTG to conduct induction. According to the scatter plots, we noticed that the cubic polynomial equation could fit the trends very well with all fitting degrees higher than 0.98.

Figure 8. Comparison graph of the fitting curves of three models.

Based on the graph where we can see several cross points’ coordinates of lines, we could adjust the IPTG concentration according to the induction time which could be pre-decided by the production plan in the future. 1.When the induction time is given less than 2.5 hours, 1 mM IPTG would be more recommended; 2.When the induction time is given during 2.5 hours 10.2 hours, 0.01 mM IPTG would be more recommended; 3.When the induction time is given during 10.2 hours ~ 19 hours, 1 mM IPTG would be more recommended; 4.When the induction time is given very enough like more than 19 hours, 0.01 mM IPTG would be more recommended.

Future plan

Testing the effectiveness of producing cellulase of positive recombinant bacteria. This is important because we need to know how much bacteria is needed to be used to effectively help animals digest fodder. In this experiment, we can control the time that positive recombinant bacteria grow and use the OD test to set the numbers of bacteria. Then put different numbers of bacteria and cellulose filter paper together. OD test can be used again to see the glucose concentration.

References

MUCK R，NADEAU E，MCALLISTEＲ T，et al． Silage review: recent advances and future uses of silage additives ［J］． J Dairy Sci，

刘海燕，张鹏举，王秀飞，等． 正交试验法优化玉米秸秆穰叶青贮发酵剂的研究 ［J］． 中国饲料，2018 ( 11) : 74－79．

Coward-Kelly G., Aiello-Mazzari C., Kim S., Granda C., and Holtzapple M., 2003, Suggested improvements to the standard filter paper assay used to measure cellulase activity,Biotechnology & Bioengineering, 82(6): 745-749.

Ghose T.K., 1987, Measurement of cellulase activities, Pure & Appl Chem, 59(2): 257-268.

Lu Z.L., Chen D., Zhang S.S., Lu Q., and Huang R.B., 2012, A Pseudomonas aeruginosa producing alkaline cellulase, China Patent, 2012103821716