Team:SMS Shenzhen/Engineering
Engineering
Overview
Based on research results, we determined to engineer LCP and laccase CotA in our project. The first phase is constructing and characterizing two basic parts, coding sequences of LCP and laccase CotA. On the basis of the first stage, the second phase purposes to improve the enzyme activity of laccase CotA by optimizing the cultural medium and preincubation conditions. With optimized results, mutated laccase CotA, resulted from rational design and directed evolution with purpose of improving substrate affinity and activity, respectively, can be tested at optimal conditions in the third phase. Consequently, three engineering cycles, "Design → Build → Test → Learn → Design...", were established to accomplish the goal of degrading the gum base.
Phase I--Construction of LCP&Laccase CotA
Design at Level I
AAs mentioned in Design, we searched for enzymes that are capable of degrading the natural and synthetic components of gum base. Considering literature research and actual experimental conditions, we ended up focusing on two enzymes, Latex clearing protein(LCP) and Laccase CotA, which are targeted at degrading two main materials of gum base, polyisoprene and EVA, respectively. Here we introduced specific principles of biodegradation.
Latex Clearing Protein(LCP)
Lcp oxidatively attacks the double bonds of polyisoprene molecules with the help of ferrous ion Fe2+ (see Fig.1) to give cleavage products that have aldehyde and keto end groups as illustrated in Fig.2. The products of oxidized polyisoprene by LCP range from C20 tetra-isoprenoid to at least C35 hepta-isoprenoid as shown in Fig.2, which indicates that LCP works like endonuclease that can cleave the substrate at different positions.
Fig.1 |The mechanism of oxydizing polyisoprene by Lcp[1]
Fig.2 |The cleavage products of polyisoprene by LCP[2] and A UV(210-nm) absorbance spectrum of LCP products(gray line)[3]
Fig.3 |The plasmid map of pET28b::LCP-K30
The plasmid integrated complete coding sequence of LCP from Streptomyces sp. K30 with a His-tag sequence at the C-terminus of LCP sequence, pET28b::LCP-K30, would be synthesized(Fig.4). We chose E.coli BL21 to express LCP, since literature supported heterologous expression in E.coli. Besides, modeling can help us predict the expression curve of LCP to predict the time for harvesting cells. Limited by experimental conditions, we couldn't follow the protocol of testing enzymes activity on the document, which qualitatively and quantitively characterize the activity of LCP. We also came up with a new measurement of testing the activity of LCP by utilizing the reaction of Schiff reagent indicating the formation of aldehyde groups. At the absorption peak of Schiff reagent, the absorbance of reaction mixture, including buffer, natural latex and LCP, should regard with the enzyme activity.
Laccase CotA
Laccase, a common oxidase, is supposed to have broad substrate specificity. As EVA is a multi polymer, its accessibility of laccase active sites is limited. This limitation is overcome by the supplementation of mediator, ABTS (2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) or 1-Hydroxybenzotriazole (HBT) as an electron carrier between laccase and substrate, as presented in Fig.4. The mediator is initially oxidized as an intermediate with high redox potential by laccase. Then, the mediator, a small chemical compound, gets access to oxidize the substrate more easily compared with enzyme. Therefore, the substrate EVA that cannot be oxidized directly by laccase is oxidized by the intermediate, while the oxidized mediator is reduced to its initial form. The mediator can maintain the cyclic redox conversion.[4] The overall oxidation system is called laccase-mediator system(LMS).
Fig.4 |The laccase-mediator system
Fig.5 |The plasmid map of pET28b-T7-laccase
The plasmid pET28b-T7-laccase including the codon-optimized complete coding sequence was obtained from iGEM20_Shanghai_SFLS_SPBS. His-tag had been already added to the C-terminus of laccase CotA. We followed their suggestions to express laccase in E.coli BL21 as well. Modeling can help to predict the time for harvestation of laccase CotA. We would conduct enzyme activity tests, according to the literature, by measuring the absorbance of reaction mixture at 420nm, which is the absorption peak of oxidized ABTS, oxidation product. This is a stable and widely-used measure of measuring the activity of laccase.
Build at Level I
LCP
We constructed a plasmid pET28b::LCP-K30, which integrated the codon-optimized complete coding sequence of Latex Clearing Protein from Streptomyces sp. K30. The original coding sequence of Latex Clearing Protein was from Genbank dataase (reference code: AY387589.1). The plasmid was transformed into E.coli BL21(DE3). The overnight culture in Luria-Bertani(LB) medium was inoculated 1:100 with fresh medium containing kanamycin and cultured at 37°C with shaking 220rpm for about 4 hours until the OD600 reaches 0.5-0.6. Then the cells were induced with 0.1mM isopropyl-b-D-thiogalactoside(IPTG) and supplemented with 8uM HemeB. Cells were cultured at 16°C/220rpm for about 16hours before harvestation.
Laccase CotA
We collaborated with iGEM20_Shanghai_SFLS_SPBS team and obtained plasmid pET28b-T7-laccase, which includes the codon-optimized coding sequence of a type of laccase from Bacillus sp. HR03, Laacase CotA. The plasmid was transformed into E.coli BL21(DE3). The overnight culture in Luria-Bertani(LB) medium was inoculated 1:100 with fresh medium containing kanamycin and incubated at 37°C with shaking 220rpm for about 4 hours until the OD600 reaches 0.5-0.6. Then the cells were induced with 0.1mM isopropyl-b-D-thiogalactoside(IPTG) and supplemented with 2mM CuSO4. Cells were cultured at 18°C/180rpm for 4 hours, and then the shaker was turned off for maintaining a microaerobic condition where laccase activity was higher according to the literature. Cells were harvested after 16h-20h by centrifugation.
Test at Level I
LCP
After being centrifuged at 4000g, 4°C, for 20 minutes, sedimented cells were resuspended in lysis buffer containing lysozyme and protease inhibitor. After an hour, cells were sonicated on ice. Disrupted cells were removed by centrifugation(10000g for 20-30 minutes at 4°C). The supernatent was utilized to confirm the success of expression when carrying out SDS-PAGE. A standard curve of formaldehyde was established to measure the number of aldehyde groups formed as a result of oxidation of polyisoprene. The assay mixture is made up of LCP, 1% natural latex solved in n-hexane, and 100 mM potassium phosphate buffer(pH 7). The mixture was incubated at room temperature for 2 hours. The LCP activity was measured by the increase in absorbance at 540nm where the absorption peak of Schiff reagent is.
Fig.6 |The SDS-PAGE gel of LCP
Fig.7 |The enzymatic activity of LCP
Laccase CotA
After being centrifuged at 4000g, 4°C, for 20 minutes, sedimented cells were resuspended in lysis buffer containing lysozyme and protease inhibitor. After an hour, cells were sonicated on ice. Disrupted cells were removed by centrifugation(10000g for 20-30 minutes at 4°C). The supernatent was utilized to confirm the success of expression when carrying out SDS-PAGE. His-tag purification resin was balanced by lysis buffer by centrfugation(1000g for 10s at 4°C). The supernatent of disrupted cells after centrifugation was added into the resin. The mixture was incubated on the platform laboratory shaker for an hour in order to allow the resin to bind with His-tag. Then, the mixture was loaded on an empty affinity chromatography column. Washing buffer was loaded on the column to remove proteins that did not specifically interact with the nickel ion. The column is then eluted with elution buffer to obtain purified protein, which was collected and concentrated by ultrafiltration (cutoff 10 kDa) later. We carried out SDS-PAGE on purified protein to confirm the success of purification. Then, the protein concentration was determined by Bradford assay using BSA as standard. Before laccase activity was measured spectrophotometrically at room temperature 27°C using ABTS as substrate, purified laccase was preincubated at 70°C for 15 minutes according to the literature. In the assay mixture, oxidation of ABTS (2 mM) in 0.1M sodium citrate buffer (pH 3) was measured by the increase in absorbance at 420 nm.
Figure 8|The activity of laccase CotA preincubated at different temperature for different periods of time.
Learn at Level I
Here we successfully heterologously expressed LCP and CotA. As expected, a significant protein expression was observed.
The unit enzyme activity of LCP (U) was defined as 1 umol of aldehyde groups produced per min. LCP activity was relatively high at room temperature.
However, we are struggling to purify the LCP. It is reported that LCP has limited solubility, which means even through we successfully expressedd them in the E.coli, it is hard to extract them from the cell. Some tag, such as NusA, can increase the protein solubility while expressing. In the future, we are planning to add those tags for the LCP to purify them. Then, the unit enzyme activity of LCP can characterize the activity of LCP more accurately.
The unit enzyme activity of CotA (U/mg) was defined as 1 umol of ABTS oxidized per min by per mg enzymes. The optimum preinucbation temperature for CotA activity was 60°C for 30 minutes, and CotA activity was relatively low at room temperature.
Phase II--Optimization of expressing and testing Laccase CotA
Design at Level II
Following previous literature research, we noticed that anion inhibition existed in the expression of fungal and bacterial laccases when exposed to chloride Cl-. Since no one had tested the effect exerted on enzyme activity by the sodium chloride in cultural medium and supplemented solution, we altered the cultural medium and supplemented solution when culturing bacteria. The settings of experimental groups and control groups are presented below.
Table 1|The setting of experimental group and control groups
We also altered the preincubation conditions of testing laccase. The enzyme were preincubated at every 10°C from 30°C to 100°C for 5/10/15 minutes before testing the enzyme activity. The data can be applied to Michaelis-Menten equation, which can demonstrate the substrate affinity of laccase.
Fig. |The sketch diagram of 96-well plates of testing preincubation conditions
Build at Level II
A single colony of E.coli carrying plasmid pET28b-T7-laccase was picked and precultured overnight in either original LB meidum containing NaCl or LB meidum containing KH2PO4. Then,the overnight culture was inoculated 1:100 with 2 types of fresh medium containing kanamycin and incubated at 37°C with shaking 220rpm for about 4 hours until the OD600 reaches 0.5-0.6. Then the cells were induced with 0.1mM isopropyl-b-D-thiogalactoside(IPTG) and supplemented with 2mM CuSO4 or 2mM CuCl2. Cells were cultured at 18°C/180rpm for 4 hours, and then the shaker was turned off. Cells were harvested after 16h-20h by centrifugation.
Test at Level II
After being centrifuged at 4000g, 4°C, for 20 minutes, sedimented cells were resuspended in lysis buffer containing lysozyme and protease inhibitor. After an hour, cells were sonicated on ice. Disrupted cells were removed by centrifugation(4000g for 20 minutes at 4°C). The supernatent was utilized to confirm the success of expression when carrying out SDS-PAGE. His-tag purification resin was balanced by lysis buffer by centrfugation(1000g for 10s at 4°C). The supernatent of disrupted cells after centrifugation was added into the resin. The mixture was incubated on the platform laboratory shaker for an hour in order to allow the resin to bind with His-tag. Then, the mixture was loaded on an empty affinity chromatography column. Washing buffer was loaded on the column to remove protein that were not specifically interact with the nickel ion. The column is then eluted with elution buffer to obtain purified protein, which was collected and concentrated by ultrafiltration (cutoff 10 kDa) later. SDS-PAGE utilized purified protein to confirm the success of purification. Then, the protein concentration was determined by Bradford assay using BSA as standard. Before laccase activity was measured spectrophotometrically at room temperature 27°C using ABTS as substrate, purified laccase was preincubated at different temperatures from 30°C to 100°C every 10°C for 5/10/15 minutes. In the assay mixture, oxidation of ABTS (2 mM) in 0.1M sodium citrate buffer (pH 3) was measured by the increase in absorbance at 420 nm.
Figure 9|The setting of experimental group and control groups
Figure 10|The activity of laccase CotA expressed in medium containing medium containing KH2PO4/NaCl and supplemented with CuCl2/CuSO4
Learn at Level II
After testing the different effects of ions, we noticed that to mass produce CotA, there are far more factors we should consider. Since phosphate group will combine with copper ions and form precipitations, laccase expressed in medium containing KH2PO4 actually cannot bind with enough copper ions. As a result, laccase CotA expressed in medium containing KH2PO4 doesn't display higher activity than normal LB medium. Besides, chloride ions in supplemented solution influence the activity as predicted. In conclusion, carbon sources, culture temperature, medium pH and mediator, those factors should be considered together and adjusted to fulfill needs of the industry.
Phase III--Improvement of Laccase CotA
Design at Level III
Based on the literature review, we utilized two approaches to improve the enzyme activity of Laccase CotA. One is rational design serving to modify the substrate affinity between Laccase CotA, of which substrate is 2,2'-azino-bis(3-ethylbenzothiazoline-6-sul-fonic acid) (ABTS), and the other one is directed evolution with the purpose of improving the thermal stability of Laccase CotA at ground temperature.
Rational Design
We carried out homology modeling to identify the probable 3D-structure of laccase CotA, molecular docking to predict the binding sites on laccase CotA, and multiple sequence alignment to verify the validity of binding sites. Then, recommended mutations, which can improve substrate affinity, were compared by molecular docking software. Among suggested single mutation, double mutations, and triple mutations, we chose three most stabilized mutations to construct parts of mutated Laccase CotA on the basis of pET28b-T7-laccase by PCR with primer resulted in site-directed mutation.
The activity test results can be applied to Michaelis-Menten equation as well. Hence, the substrate affinity of mutated laccase can be compared with that of original laccase.
Directed Evolution
We made a plan of conducting directed evolution experiments. Randomly mutated sequences coding Laccase CotA would be yielded from Error Prone-PCR(EP-PCR). Then, single colonies would be cultured to express mutated Laccase CotA. We implemented library screening for beneficial variations by testing activity of crude enzymes. Next, those colonies obtained beneficial variations would be cultured in shaken flasks. Finally, the enzyme activity results of purified mutated enzymes would be compared with those of original Laccase CotA.
Build at Level III
Rational Design
We achieved site-directed mutations on the sequence of laccase CotA by conducting PCR with primers that contain mutation, resulting fragments with desired modifications. The primers designed for 386-site mutation and 497-site mutation are shown below. After that, the linearized mutated plasmid is ligated by Gibson.
Table 2 |Primers designed for mutation
Sequence of laccase CotA with double mutations was achieved by conducting one site-direct mutagenesis PCR on the basis of the other mutated plasmid. For example, applying primers that would result in 386-site mutation on plasmid with 497-site mutation can generate the plasmid mutated at both 386-site and 497-site. After that, the linearized mutated plasmid should be ligated by Gibson as well.
Directed Evolution
With the assistance of EP-PCR kit, the coding sequence of laccase CotA should be cloned from the plasmid pET28b-T7-laccase. Because of the limitation of polymerase activity, the sequence of laccase CotA should be divided into two parts. Thus, two pairs of primers were designed and applied in the PCR for cloning sequence of laccase CotA from the plasmid. Then, error-prone PCR(EP-PCR) were separately carried out on two partial sequences of laccase CotA. We recommended that the number of cycling rounds is more than 50 times, for 30 cycling rounds have failed in cloning enough replicates of laccase CotA, while 50 and 60 cycling rounds have succeeded. Besides, after conducting EP-PCR, we have tried to ligate the plasmid backbone and two parts of randomly-mutated laccase CotA sequence by Gibson. Unfortunately, the false positive rate is so high that only 11 single colonies seemed to harbour plasmids integrating complete sequences of laccase CotA displayed by the results of colony PCR. We optimized the ligation by implementing overlap extension PCR to construct the randomly-mutated sequences of laccase CotA before ligating the complete sequences into plasmid backbones. The schematic diagram of overlap extension PCR is shown here.
The plasmid was transformed into E.coli BL21(DE3). The overnight culture in Luria-Bertani(LB) medium was inoculated 1:100 with fresh medium containing kanamycin and incubated at 37°C with shaking 220rpm for about 4 hours until the OD600 reaches 0.5-0.6. Then the cells were induced with 0.1mM isopropyl-b-D-thiogalactoside(IPTG) and supplemented with 2mM CuSO4. Cells were cultured at 18°C/180rpm for 4 hours, and then the shaker was turned off. Cells were harvested after 16h-20h by centrifugation.
Test at Level III
After being centrifuged at 4000g, 4°C, for 20 minutes, sedimented cells were resuspended in lysis buffer containing lysozyme and protease inhibitor. After an hour, cells were sonicated on ice. Disrupted cells were removed by centrifugation(4000g for 20 minutes at 4°C). The supernatent was utilized to confirm the success of expression when carrying out SDS-PAGE. His-tag purification resin was balanced by lysis buffer by centrfugation(1000g for 10s at 4°C). The supernatent of disrupted cells after centrifugation was added into the resin. The mixture was incubated on the platform laboratory shaker for an hour in order to allow the resin to bind with His-tag. Then, the mixture was loaded on an empty affinity chromatography column. Washing buffer was loaded on the column to remove protein that were not specifically interact with the nickel ion. The column is then eluted with elution buffer to obtain purified protein, which was collected and concentrated by ultrafiltration (cutoff 10 kDa) later. SDS-PAGE utilized purified protein to confirm the success of purification. Then, the protein concentration was determined by Bradford assay using BSA as standard. Before laccase activity was measured spectrophotometrically at room temperature 27°C using ABTS as substrate, purified laccase was preincubated at the optimal temperature and stimulus time derived from the results in phase II. In the assay mixture, oxidation of ABTS (2 mM) in 0.1M sodium citrate buffer (pH 3) was measured by the increase in absorbance at 420 nm.
Figure 11|The SDS-PAGE gel of original and mutated laccase CotA
Figure 12|The acitivty of original and mutated laccase CotA
Learn at Level III
Rational Design
Data derived from wetlab, the absorbance of assay mixture, can be applied to draw the diagram of Michaelis-Menten equation, which indicates the difference in substrate affinity of mutated laccase and original laccase. Besides, improvement on modeling can probably reach higher enzyme activity.
Directed Evolution
After testing the enzyme activity, analysis on the mutated laccase can be conducted. Homology modeling can predict the probable 3D-structure of mutated laccase to uncover the reason why mutated laccase can better oxidized ABTS.