Part:BBa_K4332002
Added by LZU-HS-China-C
Using cell surface display technology, the ice crystal nucleoprotein-laccase (INP-Lacc6) fusion polypeptide is first synthesized, and then transformed to anchor it on the outer cell membrane of the probiotic EcN to improve the efficiency of its degradation of antibiotics on the cell membrane surface.
We found that the laccase BBa_K729002 may also have the effect of degrading antibiotics. So we used this laccase to complete the verification experiment of degrading antibiotics.
At the same time, in order to enhance its function, we added INP protein to the N segment of BBa_K729002, so that the laccase was anchored on the surface of the bacteria through cell surface display technology.
In order to facilitate the comparison of the degradation ability of BBa_K729002 and BBa_K4332002 with INP added, we will compare and show these two parts.
(Correspondence between the gene in the picture and the gene in part：BBa_K729002——lacc6、BBa_K4332002——IL)
Selection of Expression Screening Markers for Fusion Proteins
In order to ensure that the recombinant engineered strain can finally be obtained by antibiotic resistance screening, it is necessary to identify the sensitivity of the wild strain ECN to several commonly used antibiotics. Under the same conditions, EcN was inoculated by the test tube culture method to a test tube containing a final concentration of 30 μg/ml chloramphenicol, 50 μg/ml ampicillin, and 50 μg/ml kanamycin. Wild-type EcN can't survive in the antibiotics containing the above several kinds of clones, so it can be used as a host bacteria to express the fusion protein.
Construction of expression vector
In order to obtain a high-copy constitutive plasmid, the recombinant plasmid pSB1A3-mRFP derived from IGEM tissue was modified. Under the action of specific primers, the rfp gene sequence on it was successfully removed from the plasmid DNA sequence, and NdeI, BamHI and HindIII restriction sites were introduced. For application, pSB1A3-mRFP was first linearized by BKL kit (Takala, Japan), rfp sequence was removed, restriction site was introduced, and then the plasmid was recircularized under the action of Blunting Kination Enzyme and named pSB18A. Finally, the recombinant plasmid pSB18A/INP-N-L was successfully transformed into the expression strain EcN and named EcN-IL.
Determination of the expression position of the fusion protein
Cell fractionation was used to analyze the subcellular localization of INP-N-Lacc6 fusion protein expression. The size is 104 KDa, and no fusion protein band was detected in the intracellular membrane component of EcN. a. After western blotting, it was found that the fusion protein was found in the total bacterial protein lysate (T) and the outer cell membrane fraction (OM) obtained by centrifugation, while the inner cell membrane fraction (IM) was found. No obvious bands were found in the periplasmic component (S). The experiment found that certain bands also appeared in the periplasmic component (S). Therefore, it is speculated that the INP-N-Lacc6 fusion protein may be synthesized in the body first, and then anchored on the cell membrane surface. During the experiment, EcN carrying empty pSB18A was used as a control, and it was found that there was no expression of fusion protein in the cytoplasm, inner cell membrane, and outer cell membrane components.
Analysis for subcellular location of expressed INP-N-lacc6 fusion protein in EcN. (a) SDS-PAGE analysis. M: Protein marker 3451, Lin2,4,6,8: EcN-IL, Lin1,3,5,7: EcN- pSB18a, (b) Western bloting analysis of INP-N-Lacc6 in total cell lysate(T), soluble fraction(S), outer membrane(OM), and inner membrane(IM) was detected by Cell fractionation. Arrow indicates the location of the fusion proCtein. Cells harboring pSB18a were used as negative control.
Analysis of qRT-PCR detection results
In order to verify the relative expression of the lacc6 gene in the engineered strains, different control strains were added in this study, such as wild EcN, carrying empty EcN (EcN-pSB18a). In order to prove that the expression of the lacc6 gene is not affected by the INP-N protein, an engineered strain with intracellular expression of lacc6 was constructed as a control and named EcN-Lacc6.
After analysis, it can be seen that the expression level of the lacc6 gene in EcN-Lacc6 after modification is about 9 times higher than that of wild-type EcN and EcN-pSB18a carrying an empty vector. The expression level of the lacc6 gene in EcN-IL is about 10-fold higher than that of wild-type EcN.
Enzyme activity of engineering strains and determination of optimal reaction conditions
In order to determine the enzymatic activity of whole-cell catalysts, this experiment uses 2,2-diazo-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) as the substrate, and the absorption wavelength is 480 Calculate its enzyme activity under nm conditions. (Bebrone, 2007). First, the engineered strains were inoculated into LB medium at 37°C overnight with 1% inoculum. After centrifugation to remove the supernatant, the bacteria were collected (6000 r/min, 10 min). The bacteria were washed twice with an equal volume of PBS (pH=7.0) buffer and prepared to become bacteria with a cell concentration of 1.0×109 CFU/mL The suspension is ready for use. The reaction temperature is set to 30°C, the enzyme activity reaction system is 3 mL, which contains 1 mL of 5 mmol/L ABT, 0.5 mL of bacterial cell suspension, and 1.5 mL of 0.1 mol/L malonate buffer (PH=5.0). 1 unit of enzyme activity (U) refers to the amount of ABTS that can oxidize 1 mmoL of ABTS within 1 minute by whole-cell catalyst at 30°C to increase the product of ABTS free radicals from a to b. The unit of enzyme activity is 1 mmoL/min, That is U/g (solid).
The laccase enzyme activity of the whole-cell catalyst was detected to be 1.99 ± 087 U/g dry cell weight. However, the enzyme activity was not detected in the control group EcN-pSB18a and wild-type EcN. This result indicates that the laccase gene was successfully expressed on the cell membrane and exhibited high enzyme activity. The experiment was set to three replicates.
In order to detect the optimal pH of the reaction, the engineered strain was first inoculated into LB medium at 37℃, 180 r/min, 10min overnight culture with 1% inoculum. After centrifugation to remove the supernatant, collect the bacteria (6000 r/min, 10min), suspend the bacteria in an equal volume of PBS buffer with pH=4, 5, 6, 7, 8, 9, and then in each system Sulfadiazine sodium salt was added at a concentration of 50 mg/L. After reacting for 1 h, the bacterial pellet was discarded by centrifugation, and the residual amount of SDZ in the supernatant was detected by HPLC. In order to determine the optimal reaction temperature, after obtaining the bacterial cells with the same treatment method, they were suspended in an equal volume of PBS buffer at a temperature of 25, 30, 35, 40, 45, and 50 ℃, and the same was added to it. Sulfadiazine sodium salt was added at a concentration of 50 mg/L. After 1 h of reaction, the supernatant sample was centrifuged and filtered through a 0.22 μm filter membrane. The degradation of SDZ by EcN-IL was detected by HPLC.
When the pH is in the range of 4 to 7, the degradation rate of SDZ by EcN gradually increases, and the degradation rate reaches the maximum at pH=7.0. As the pH gradually increases, the degradation efficiency gradually decreases; in Figure 3-2 At the same time, it is concluded that the degradation rate of SDZ gradually increases with the increase of temperature, and its degradation efficiency is the highest when the temperature is 40℃. Therefore, the optimum temperature for the reaction is 40°C and the optimum pH is 7.0.
The ability of fungal laccase to degrade sulfadiazine
EcN-Lacc6 refers to the fungal laccase gene expressed in the cell
This experiment mainly uses HPLC to quantitatively analyze the residues of sulfadiazine. The process involves two parts: sample pretreatment and HPLC on-machine detection. The specific experimental method is as follows;
(1) Sample pretreatment. First, EcN-IL and the control wild-type EcN, the unloaded EcN and the intracellular expression strain EcN-Lacc6 were inoculated into LB medium at 37°C overnight at an inoculum of 1%. After centrifugation to remove the supernatant, the bacteria were collected (6000 rpm, 10min). The bacteria were washed twice with the same amount of PBS (pH=7.0) buffer solution and prepared into a bacterial suspension with a cell concentration of 1.0 × 109 CFU/mL. Take 10 mL of the cell suspension and add SDZ sodium salt with a concentration of 30, 50, and 100 mg/L into it, with three replicates in each group. Incubate at room temperature for 3 hours and then centrifuge (10000 r/min, 10 min), collect the supernatant into a 10mL PE tube, and finally filter it into a special brown sample bottle for HPLC using a 0.22μm filter membrane.
(2) HPLC on-machine detection conditions selection. HPLC loading conditions selection: select VWD detector, detection wavelength is set to 270 nm, methanol and ultrapure water are organic phase, mobile phase: methanol: water=25:75, detection column Waters XTERRA MS C18 column (250× 4.6 mm), the column temperature is set to 30°C, and the mobile phase flow rate is 1.0 mL/min. HPLC detection data is calculated using external standard method.
ice crystal nucleoprotein-laccase (INP-Lacc6)
Using cell surface display technology, the ice crystal nucleoprotein-laccase (INP-Lacc6) fusion polypeptide is first synthesized, and then transformed to anchor it on the outer cell membrane of the probiotic EcN to improve the efficiency of its degradation of antibiotics on the cell membrane surface.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 72
Illegal NgoMIV site found at 405
Illegal NgoMIV site found at 1155
Illegal AgeI site found at 823 - 1000COMPATIBLE WITH RFC[1000]
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