Team:Edinburgh OG/Immobilisation

Enzyme Immobilisation

We are exploring enzyme immobilization as bioremediation delivery systems. The benefit of immobilizing dye-degrading enzymes relies in the potential to increase enzymatic lifespan while preserving bioactivity. As well, in some cases as the biochar immobilization, avoiding the release of genetically modified microorganism into the environment.

Azo-reductase extracellular immobilization

The very first step in azo dye degradation is the cleavage of the nitrogen double bond. It is considered chemically stable but can be biologically broken by an enzyme called azoreductase. The reductive cleavage of nitrogen double bond normally consists of two steps that both requires NAD(P)H as electron donor. (Figure X) Among all the studies related to azoreductases, one the one from Klebsiella Oxytoca is the most suitable one for immobilization since its oxygen insensitive and can function without flavin cofactor. (Hua and Yu, 2019) Immobilizing azoreductase on E. coli extracellular matrix could be a strong candidate to enhance enzyme stability while solve the mass transportation issue since most azoreductases are cytosolic. Curli fiber is an important component of extracellular material that protect cell from environmental stresses. Its broad surface area makes it a perfect platform for enzyme immobilization. Biofilm Integrated Nanofiber Display (BIND) is a novel approach that fuses heterologous functional protein domains to curli fiber monomer protein CsgA. (Botyanszki et al., 2015) (Figure X) A SpyTag/SpyCatcher system is adopted in this approach to form an irreversible conjugation between curli and enzyme.

Figure 1. Azoreductase catalysis: reductive cleavage of the nitrogen double bond in Methyl Red molecule. The reaction consists of two steps that both requires NAD(P)H as electron donor. The final products of the reaction are N, N-Dimethyl p-phenylenedianmine and o- aminobenzoic acid (Hua and Yu, 2019).

Figure 2. Catalytic-BIND: the display of an enzyme on E. coli curli fibers, resulting in the functionalization of biofilms. (A) Genetically modified E.coli that is capable of expressing CsgA fused to SpyTag (CsgA-ST). CsgA-ST self-assembles into curli fibers on the surface of the bacterium. During the process of biofilm formation, curli fibers can create a polymer matrix surrounding cells. (B) The polymer matrix is covalently conjugated with an enzyme fused to a SpyCatcher (enzyme-SC). (C) Substrate to product transformation occurs on the high-surface area of the biofilm (Botyanszki et al., 2015).

Design

Two types of strains are designed to perform the job. The curli producing strain is responsible for CsgA-SpyTag curli fiber producing while the protein expressing strain is responsible for Enzyme-SpyCatcher producing. Both gene circuits are designed to be producing corresponding protein under an inducible manner, as a proof of concept that the immobilization will spontaneously occur when the dye sensor developed in this project sense the dye.

The curli producing strain is E. coli PHL628 - ΔcsgA (MG1655 malA-Kan ompR234 ΔcsgA). It was provided by The Joshi Lab at Harvard University. The CsgA gene in the genome of this strain is deleted. pBbE1a-CsgA-25AASpyTag is responsible for inducible synthetic curli production. The protein expressing strain is responsible for azoreductase-SpyCatcher production prior conjugation. Native azoreductase was used to set up the base line of enzyme activity while the sfGFP-SpyCatcher was used as a visual aid of the conjugation. They are expressed in separate strains. The chassis used for protein production is BL21(DE3) while the backbone is pET28a-gg. The gene constructs involved in this bit are listed in Figure 3.

Figure 3. The schematic diagram for four gene constructs assembled in this study. Colorful arrows indicate the gene fragments, with the arrow indicating the orientation of each component. (A) pET28a-AzoR. It was used in inducible AzoR production. (B) pET28a- gg-AzoR-SC. It was used in inducible AzoR-SC production. (C) pET28a-gg-sfGFP- SC. It was used in inducible sfGFP-SC expression. (D) pBbE1a-CsgA-25AAST.The SpyTag used in this study is 25AA-SpyTag developed by Botyanszki et al. It was used in inducible curli production.

Experiments

The construction of expression vectors

Since the four constructs were all designed to be inducible expression by IPTG, all plasmids involved have IPTG inducible promotor. The curli producing construct was assembled using enzyme digestion/ligation method while the three protein expression constructs were assembled using golden gate method. The amplified inserts including AzoR, AzoR-SpyCatcher (AzoR-SC) and sfGFP-SpyCatcher (sfGFP-SC) were assembled into vector pET28a-gg-mCherry using golden gate reaction. One reaction of golden gate contains 100ng of vector DNA, insert DNA 30ng, 2x T4 ligase buffer 2μL, BsaI 1μL, T4 DNA ligase 1μL and nuclease-free water to 20μL. The thermocycling conditions for golden gate reaction was: 37°C 1.5min, 16°C 3min repeated for 25 cycles, 50°C 5min, 80°C 10 min and 4°C hold. The curli producing construct was built by digestion and ligation. The digestion to acquire the backbone of pBbE1a-RFP was conducted in a final volume of 200μL including 20μL of New England BioLabs (NEB) 10x Cutsmart buffer, 1μL EcoRI-HF, 1μL BamhI-HF and 1μg plasmid DNA. The digestion of CsgA-25AAST gene fragment was carried out in a final volume of 100uL containing 10μL of New England BioLabs (NEB) 10x Cutsmart buffer, 1μL EcoRI-HF, 1μL BamhI-HF and 100ng amplified insert DNA. Ligation was a 20μL reaction containing T4 DNA ligase 1μL, 2xT4 ligase buffer and the ratio of insert DNA to backbone DNA was 10:1 (calculated with NEB ligation calculator).

The inducible production of recombinant protein

For recombinant protein expression, BL21(DE3) cells transformed with pET28a-gg based constructs were grown overnight in LB medium at 37°C with 100μg/mL kanamycin. The overnight culture was then 1:100 diluted, and then incubated at 30°C until OD600 of 0.4 was reached. The expression of three recombinant proteins were induced with 0.5mM final concentration of IPTG and shaken overnight at 18°C. As for the recombinant curli expression, PHL628-ΔcsgA cells were transformed with pBbE1a-CsgA-25AAST. The cells were streaked onto YESCA plates with 100μg/mL ampicillin. Transformed cells were collected, grouped and inoculated in YESCA medium with the same concentration of ampicillin until an OD600 of 0.4 was reached. Curli expression was induced with 0.3mM final concentration of IPTG and shaken 18-24h at 25°C (Botyanszki et al., 2015; Hua and Yu, 2019).

SDS-PAGE was used to verify the inducible expression of four proteins. Induced and uninduced cell cultures were spun down and resuspended in 200μL 1x SDS Sample Buffer (100μL 2x Sample Buffer contains 95μL 2x Laemmli Sample Buffer from Bio-Rad and 5μL βmercaptoethanol). Supernatants of cell cultures were mixed 1:1 with 1x SDS Sample Buffer. The samples were then boiled for 3min at 96°C. 10μL of each sample was loaded per well in Mini-Protein® TGXTM Precast Gel produced by Bio-Rad. Electrophoresis was run at 200V for 20 to 30min. InstantBlueTM purchased from Sigma-Aldrich was used to stain SDS-PAGE gels overnight.

The colorimetric assay of recombinant azoreductase

To test enzyme activity, the cultures were either spun down to acquire supernatant samples or sonicated to acquire cell lysate samples. A Soniprep 150 sonicator developed by MSE was used. Each cell culture was treated at 230kHz for 30s and the rest for 30s for three times. To test azoreductase activity against specific dyes, a standard enzyme assay contained, in 900μL PBS (pH7.0): 450μmol NADH, 100nmol MR or 50nmol RB5 and 300μL of supernatant or cell lysate. The reactions were spectrophotometrically assayed at room temperature at 420nm (ε=24.96mM-1cm-1) for MR and 597nm (ε=21.03mM-1cm-1) for RB5. The reactions were started by the addition of NADH solutions. Each unit of azoreductase activity (U) was defined as the amount of enzyme that catalyzed decolorization of 1mmol of azo dye per minute (Hua and Yu, 2019). Each reaction was carried out in triplicate, a student’s t test was used to determine the significance between experimental group and control groups.

Congo red binding assay of induced curli fiber

Congo Red (CR) binding assay was adopted to measure curli production. 1mL of induced liquid culture was pelleted at 5000g for 10min and resuspend in PBS. Congo Red was added to the resuspended cell culture at 25μM and incubated at 25°C for 10 min. Cells were then pelleted at 21000g and the absorbance of the supernatant was measured at 490nm. The quantity of CR binding was determined as the subtraction of the above measurement by a CR+PBS control (Botyanszki et al., 2015). Each reaction was carried out in triplicate, a student’s t test was used to determine the significance between experimental group and control groups.

Results

Cloning

We successfully assembled four constructs designed in this part of our project, pET28a-gg-AzoR, pET28a-gg-AzoR-SC, pET28a-gg-sfGFP and pBbE1a-CsgA-25AAST. They were verified by diagnostic restriction enzyme digestion shown in Figure 4.

Figure 4. Verification of the assemly using restriction enzyme (RE) digestion. 1 kb ladder from NEB was used to run a 1% agarose gel. The samples in lane 1 to lane 13 are: pET28a-gg-AzoR cut by BstEII, pET28a-gg-AzoR uncut, pET28a-gg-AzoR-SC cut by PvuI, pET28a-gg-AzoR-SC uncut, pET28a-gg-sfGFP cut by PvuI, pET28a-gg-sfGFP uncut, pBbE1a-CsgA-25AAST cut by BstEII, pBbE1a-CsgA-25AAST uncut, pET28a-gg-mCherry cut by BstEII, pET28a-gg-mCherry cut by PvuI, pET28a-gg-mCherry uncut, pBbE1a-RFP cut by BstEII and pBbE1a-RFP uncut, respectively. Some digestions were not complete, nevertheless, bands corresponding to the four gene constructs distinguished them from that of the vectors. Successful insertion was verified.

Inducible Expression

The inducible expression result was verified by SDS-PAGE. The inducible expression of AzoR-SC and sfGFP-SC were successful while the other two failed. The results were shown in Figure 5.

Figure 5. SDS-PAGE results of the induction of 4 constructs. Cell pellets were used for samples. PageRulerTM Plus Prestained Protein Ladder was used. Samples for lane 1 to lane 8 are: AzoR induced, AzoR uninduced, AzoR-SC induced, AzoR-SC uninduced, sfGFP-SC induced, sfGFP-SC uninduced, CsgA-25AAST induced and CsgA25AAST uninduced, respectively. Plus indicates the sample is induced, while minus indicates the sample is uninduced. The circled bands are the induced protein band for each sample Induction was successful for AzoR-SC and sfGFP-SC at desired size (37kDa, 42kDa). The expressed AzoR was not corresponding with desired size (22kDa).

Enzymatic Assay

The activity of recombinant AzoR and AzoR-SC was analyzed against two substrates, Methyl Red (MR) and Reactive Black 5 (RB5). The enzyme activity of the supernatant and cell lysate were both analyzed spectrophotometrically and plotted in Figure X. A Student’s t test was adopted to determine if uninduced samples were significantly different from its corresponding induced samples. According to the bar chart, there were azorductase activity detected in wild type BL21 samples and uninduced control samples that might be due to other oxidases or reductases produced by cells. However, they are all below 5U/mL. The enzyme activity in supernatant against two substrates for neither recombinant AzoR or AzoR-SC reached significance level, indicating the enzyme was not secreted. It is notable that the AzoR-SC enzyme activity in cell lysate against MR and RB5 both reached different significant level. This indicates a functional enzyme production of recombinant AzoR-SC. However, the recombinant AzoR activity in cell lysate did not reach significant against both substrates. It is clear that the produced AzoR was inactive. Besides, the activity of cell lysate AzoR-SC against MR is 700 times higher than that against RB5. Interestingly, according to the original study that reported AzoR activity, the wild type enzyme activity against MR is 8 times of that against RB5 (Hua and Yu, 2019). These new results could mean that the specificity of AzoR against substrate MR was enhanced when linked to SpyCatcher. However, there more evidence is needed to prove this conclusion.

Figure 6. Decolorization of dye by recombinant AzoR and AzoR-SpyCatcher. Unconcentrated crude cell lysate or supernatant were used as samples for each assay. The enzyme unit was described as unit per milliliter of sample. The error bars showed the standard error for triplicates. Wild type BL21 cell culture was used in all assays as negative control. In t test, single star indicates the induced sample reached a significance of p<0.05, triple star indicates the induced sample reached a significance of p<0.01. (A) The AzoR activity against MR in cell culture supernatant, induced and uninduced. Although AzoR-SC activity was the highest among all samples, it did not reach significant level. (B) The AzoR-SC activity against MR in crude cell lysate of cell culture, induced and uninduced. The enzyme activity of AzoR-SC reached 5000U/mL and it reached the significance of p<0.01.(C) The AzoR activity against RB5 in cell culture supernatant, induced and uninduced. The detected activity from induced samples were not significantly different form their corresponding control. (D) The AzoR-SC activity against RB5 in crude cell lysate of cell culture, induced and uninduced. The AzoR-SC activity reached the significance of p<0.05.

Conclusion and Future Directions

We successfully designed and assembled four gene constructs for the establishment of the catalytic-BIND as a system to immobilize AzoR azoreductase. The constructs with enzyme expression showed excellent inducibility. Two of the recombinant proteins – AzoR-SC and sfGFP-SC – succeeded to express and the former one appeared to be high in enzyme activity. AzoR-SC activity from crude cell lysis could reach 5000 U/mL against substrate Methyl Red. The inducible expression of curli fiber failed due to poorly optimized induction conditions.

The suitability of this catalytic-BIND technology for solving the dye waste treatment issue is questionable, since the cost associated with the establishment of the system and its maintenance are both high. However, it explored a possible way to solve the dye waste treatment issue. The result of this project confirmed the conclusion of us talking to local industry that cell-free system could be a potentially better approach to combat the dye waste treatment issue. It inspired us to develop a cell-free enzyme immobilization approach.

References

Botyanszki, Z. et al. (2015) ‘Engineered catalytic biofilms: Site-specific enzyme immobilization onto E. coli curli nanofibers.’, Biotechnology and bioengineering. United States, 112(10), pp. 2016–2024. doi: 10.1002/bit.25638. Hua, J.-Q. and Yu, L. (2019) ‘Cloning and characterization of a Flavin-free oxygen- insensitive azoreductase from Klebsiella oxytoca GS-4-08.’, Biotechnology letters.

Peroxidase Ghost Shells

Anthraquinone dyes are the second-largest family of dyes used in the textile industry [1]. Anthraquinone dyes, present a higher toxicity than azo dyes, and are considered the most toxic molecules in the waste effluents from textile colorization [2]. Anthraquinone dyes may be degraded by several dye-degrading peroxidases (DyPs) [3] [4]. However, these dyes as substrates, are toxic to the cells and the use of purified DyPs results expensive procedures and costs [4]. Although the peroxidase from Bacillus subtilis (BsDyP) has been previously characterised and showed it is a promising candidate for degrading synthetic dyes [4], the one-step in situ immobilisation of this enzyme has not been explored yet. This could be an effective method to lower bioremediation costs and the levels of toxic by-products during the procedure.

We attempted to create peroxidase 'ghost shells' by immobilising the enzymes to the inner cell membrane and using lysis proteins following the one-step in situ immobilization approach proposed by Sührer et al., [5] (Fig. 1).

Fig. 1. Ghost shell diagram. The recombinant enzymes are produced by anchoring the target enzymes to the surface of the bacterial inner membrane. 2. Lysis protein E is expressed in the cytosolic plasma of the bacteria. 3. Lysis pores are generated via the fusion of the inner membrane and outer membrane under the effect of protein E, leading to the release of cytoplasm. 4. An empty cellular envelope is produced with a lysis pore that allows substrates entry and immobilized enzymes [5].

Design

In order to create the ghost shell system, two different constructs (Fusion protein DyP-Cytb5 and Lysis E) in two independent plasmids (pSB1C3-RFP and pET28a-GG) were attempted to be expressed in E. coli by double transformation.

For immobilizing DyPs from Bacillus subtilis to the inner surface of the E. coli cytosolic membrane, we created a fusion protein using the C‐terminal membrane anchor protein originating from cytochrome b5 (Cytb5). To create the ghost shells, we used the Lysis protein E from PhiX174 developed by the iGEM ETH Zurich team 2017. We hypothesized that the empty cellular envelopes with immobilized DyP could increase the bioactivity and enzymatic lifespan of peroxidases for degrading anthraquinone dyes.

To construct the fusion protein DyP-Cytb5, the first 45 amino acids sequence of BsDyP was removed according to the result of the signal peptide for DyP, which was predicted using SignalP-5.0 (http://www.cbs.dtu.dk/services/SignalP/). Then DyP was fused with C-terminal membrane anchor protein cytochrome b5 (Cytb5). The resulting sequence was labelled “DyP1”. The C-terminal anchor sequence from cytochrome b5 of rabbit liver was described in the previous report [5]. To express the secreted DyP (without C‐terminal membrane anchor), the original nucleic acid sequence with signal peptide of BsDyP was used as the coding sequence and labelled as “DyP”. For these two above constructs, the restriction sites of EcoRI and SpeI were incorporated on the 5’ and 3’ ends of the inserts respectively as they can be used clone expression cassettes within pSB1C3. The resulting of these two plasmids are shown in Fig 2.

Figure. 2. Overview of pSB1C3 with DyP-Cytb5 and DyP constructs. A: Structure of fully assembled pSB1C3 with DyP-Cytb5. B: Structure of fully assembled pSB1C3 with DyP. The plasmid contains an Origin of replication (Ori), a T0 terminator (term), chloramphenicol resistance, an RFP sequence with RBS and promoter. VF2 and VR were the primers binding sites for colony PCR.  Each construct was digested by EcoRI and SpeI restriction enzymes and then inserted into the pSB1C3 backbone to produce recombinant pSB1C3: DyP-Cytb5 and pSB1C3: DyP. 

Construct design of Lysis E 

The sequence for lysis E originates from the work of iGEM 2017 ETH Zurich team, who presented a Lysis E encoded by Phage Phi X 174 fused with an engineered RBS. The pET28a-GG plasmid was used to cloned, which contained a T7 promoter and a Lac operator binding site to control the expression of the recombinant gene and a ribosome binding site. The coding sequence of the lysis gene was named as “Lysis E”, where the RBS sequence was removed (Fig. 3)

. 

Figure. 3. Layout of the Lysis E construct, where “A” and “B” indicates BsaI recognition sites beside the ligation sites ‘A’ and ‘B’ at the 5’ and 3’ end, “S” indicates the start codon ATG, “Lysis E” indicates the coding sequence of lysis E encoded by Phage Phi X 174, “T” indicates the stop codon TAA. 

Experiments

Dye-degrading peroxidase construct - pSB1C3 plasmid

The DNA sequences of DyP and DyP-Cytb5 provided by IDT were amplified using the forward primer DyP-For and reverse primers DyP-Rev or DyP-cytb5-Rev, respectively, at Tm = 58 °C. Subsequently, purified DyP and DyP-Cytb5 fragments were cloned into the pSB1C3 plasmid and transformed into Top10 cells. Plates were grown in chloramphenicol antibiotic. Positive colonies were selected and screened by colony PCR (VF2 and VR primers, to confirm successful insertion). Then pSB-D and pSB-DC plasmids were both digested with PvuI, KasI and EcoRI.

DNA constructs were digested in 100 µl containing 450 ng DNA, 1 µl of each selected restriction enzyme (SpeI-HF and EcoRI-HF, NEB), 10 µl of 10 X CutSmart buffer at 37 °C in a water bath for two hours. Subsequently, the digested samples were purified via the QIAquick Gel Extraction Kit (QIAGEN) to eliminate the redundant restriction enzymes. The purification process followed the instructions provided by the manufacturer with the following modifications to the protocol: considered 100 ul of the digestion product via enzymatic reaction as 1 volume of gel. The ligations of the digested products were conducted in 20 µl (as described in Protocol 4).

Lysis E construct – pET28a-GG plasmid

For Golden gate colony, restriction/ligations were set up with 1 µl of each plasmid and constructed in 20 µl containing 2 µl 20 X ligase buffer (NEB), 1 µl selected restriction enzyme (BsaI, NEB) and 1 µl T4 DNA ligase (NEB). Meanwhile, three control groups were set up using water to replace (1) insert; (2) BsaI; (3) T4 DNA ligase. The reactions were incubated following the 4-step incubation: (1) 37°C for 1.5 hours (2) 16 °C for 3 minutes; (3) 50°C for 5 minutes; (4) 80°C for 10 minutes.

Induced Protein Expression

The overexpression of proteins was conducted in BL21 (DE3) host cells with an overnight incubation at 37 °C overnight. The recombinant strains were grown in LB medium, which was supplemented with necessary antibiotics at 37 °C. Then BL21 (DE3) cells expressing BsDyP and Lysis E were diluted 1000 times with fresh medium containing necessary antibiotics. The expression of Lysis E was induced by Isopropyl -β-D-1-thiogalactopyranoside (0.1 mM, IPTG, Sigma-Aldrich) in liquid culture medium. The optical density of the medium at OD600 was measured to estimate the cell density.

Cells expressing isolated DyP were cultured until OD600 value reached 0.6, followed by addition of hemin (15 µM, Sigma-Aldrich) as a co-factor to induce the overnight expression of DyP at 18 °C.

Double transformation 

Each recombinant plasmid was obtained as described in transformation protocol, which was then diluted to 10 ng/µl. The diluted plasmid (1 µl) were transformed into chemically competent BL21 cells (100 µl) and other cells as described before [6]. 

ABTS assay 

Secreted DyP cells were prepared by centrifugation at 4000 g for 10 min, followed by resuspension with potassium phosphate buffer (100 mM, pH 6.5) containing 1% Triton. The supernatant of extracted cells was harvested after centrifugation. To conduct the oxidation test of 2,2′-azino bis (3-ethylbenzthiazoline-6-sulfonic acid; ABTS), ABTS (2.0 mM, Sigma-Aldrich), H2O2 (0.2 mM, Sigma-Aldrich) and 0.2 ml supernatant of extracted cells were added to pH 4 acetate buffer (20 mM) in a total volume of 1 ml. Two control groups were set up by using water and intact cells to replace extracted cells. The enzymatic activity was measured at 420 nm (ε=36,000 M−1 cm−1) via a UV-spectrophotometer in triplicate. 

Results

Cloning

We successfully assembled pSB1C3: DyP-Cytb5, pSB1C3: DyP, pET28a-GG: Lysis E plasmid and transformed it into BL21 E. coli cells. These were confirmed by both colony PCR (Figure 4 and 5) and plasmid digestion (Figure 6 and 7).

Figure 4: Colony PCR of each eight colonies from the positive plates. PCR was conducted with colony primers at Tm = 55 °C. The results of Colony PCR were shown that DyP and DyP-Cytb5 were successfully inserted into a few positive colonies, around 1.6 kb indicating a successful insertion.

Figure 5. Digestion of pSB1C3: Dyp-Cytb5 and pSB1C3 :DyP plasmids. The digestions of the recombinant plasmids using PvuI, kasI and EcoRI suggest successful insertion.

Figure 6. Colony PCR of four pET28aGG: Lysis E colonies from positive plates. Colony PCR was conducted using T7-F and T7-R primers at Tm = 45 °C.  The bands around 467 bp indicating the successful insertion of the Lysis E into pET28a-GG was in the positive colonies. The positive control (pET28a-GG) with a band around 1 kb as expected.

Figure 7. The recombinant plasmids were digested with EcoRV, PstI and BsaI, where the target bands were observed roughly. As the bands were not clear, other gels have done again but unfortunately did not saved.

Expression analysis of DyP, DyP-Cytb5 and Lysis E 

The SDS-PAGE analysis of the crude extract cells of peroxidases shows that the supplement of hemin in the culture medium facilitated the formation of a stronger band at around 45 kDa for both DyP and DyP-Cytb5 (Figure 8). However, similar bands were also shown in cell crude extracts without hemin induction and some bands were also disappearing upon induction. Unfortunately, the SDS-PAGE analysis was not repeated to confirm the protein expression due to the time limit.

Figure 8. Protein expression of DyP and DyP-Cytb5 examined via SDS-PAGE. Visible protein bands at 45 kDa are shown in the image.

The SDS-PAGE gel was used to analyze the crude extracts of Lysis E induced with IPTG and non-induced as control but failed. Furthermore, OD600 was measured each hour to monitor the cell growth after adding IPTG. The Result shows the cell growth of BL21 (DE3) cells harboring pET28a-GG: Lysis E did not decrease (Figure 9), possibly cells did not die.

Figure 9. The growth kinetics of the E. coli empty shell harboring the pET-LE plasmid. Till the mid-log phase indicated by an arrow and OD600 reaches to 0.4, cells growing at 37°C was split in two cultures. The first was under the induction of 1 mM IPTG, while the second was the non-induced control. Cell growth was monitored through the measurement of the OD600 of the culture every hour. .

Production of ghost shells

The plasmids pET28a-GG: Lysis E and pSB1C3: DyP-Cytb5 were transformed into BL21 (DE3) cells and incubated on the plates with both chloramphenicol and kanamycin. Only pSB1C3-RFP grown on the plate with chloramphenicol and pET28a-GG plasmids grown on the plate with chloramphenicol or kanamycin displayed pink colonies while no colonies were found on other plates (data not shown), indicating successful transformation process and there might be leakage of the expression system of pET28a-GG. In conclusion, producing empty cellular envelopes (ghost shells) with immobilized peroxidase was not achieved on this project.

Conclusion and Future Directions

We successfully designed and assembled pSB1C3: DyP-Cytb5, pSB1C3: DyP, pET28a-GG: Lysis E plasmid and transformed into BL21 E. coli cells. For characterization of peroxidase, only crude extract cells of peroxidases were adopted in this project, which is difficult to determine whether the extracellular peroxidase DyP was fully expressed by using SDS-PAGE analysis. This shall be done to analyze whether the protein is expressed in intact cells, cytoplasm and membrane separately. Also, an N-terminal His-tag could assist in confirming whether the desired proteins exist on the SDS-PAGE gel. The OD600nm result of induced Lysis E by IPTG showed cells did not die, possibly due to leakage of the expression system (pET28a-GG). Troubleshooting can consider use of thermo-controlled expression system to output bacterial ghost shells [1]. Therefore, the significant steps of expressing ghost shells needs to find a stable expression system for Lysis E.

References

[1] N. A. Valdez-Cruz, L. Caspeta, N. O. Pérez, et al., “Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pL and/or pR promoters,” Microbial cell factories, vol. 9, no. 1, pp. 18, 2010.