A dual fluorescence protein expression system detects cell cycle dependent protein noise.

Inherently identical cells exhibit significant phenotypic variation. It can be essential for many biological processes and is known to arise from stochastic, 'noisy', gene expression that is determined by intrinsic and extrinsic components. It is now obvious that the noise varies as a function of inducer concentration. However, its fluctuation over the cell cycle is limited. Applying dual colour fluorescence protein reporter system, Cyan Fluorescent Protein (CFP) and Yellow fluorescent protein (YFP) tagged multi-copy plasmids, we determine variation of the noise components over the phases in lac promoter induced by Isopropyl ß-D-1-thiogalactopyranoside (IPTG) and in presence of additional Magnesium, Mg2+ ion. We, also, estimate the how such system deviates from observations of single-copy plasmid. Found 25 % difference between multi-copy system and single-copy system clarifies that observed noise is considerable and estimates population behaviour during the cell cycle. We show that total variation in cells induced with IPTG is determined by higher extrinsic than intrinsic noise. It increases from Lag to Exponential phase and decreases from Retardation to Stationary phase. By observing slow and fast dividing cells, we show that 5 mM Mg2+ increases population homogeneity compared to 2.5 mM Mg2+ in the environment. The experimental data obtained using dual colour fluorescence protein reporter system demonstrates that protein expression noise, depending on intra cellular ionic concentration, is tightly controlled by phase of the cell.

Expression and purification of active shikimate dehydrogenase from Plasmodium falciparum.

Plasmodium falciparum is known to cause severe malaria, current treatment consists in artemisinin-based combination therapy, but resistance can lead to treatment failure. Knowledge concerning P. falciparum essential proteins can be used for searching new antimalarials, among these a potential candidate is shikimate dehydrogenase (SDH), an enzyme part of the shikimate pathway which is responsible for producing endogenous aromatic amino acids. SDH from P. falciparum (PfSDH) is unexplored by the scientific community, therefore, this study aims to establish the first protocol for active PfSDH expression. Putative PfSDH nucleotide sequence was used to construct an optimized expression vector pET28a+PfSDH inserted in E. coli BL21(DE3). As a result, optimal expression conditions were acquired by varying IPTG and temperature through time. Western Blot analysis was applied to verify appropriate PfSDH expression, solubilization and purification started with lysis followed by two-steps IMAC purification. Enzyme activity was measured spectrophotometrically by NADPH oxidation, optimal PfSDH expression occur at 0.1 mM IPTG for 48 hours growing at 37 °C and shaking at 200 rpm, recombinant PfSDH obtained after purification was soluble, pure and its physiological catalysis was confirmed. Thus, this study describes the first protocol for heterologous expression of PfSDH in soluble and active form.

Mechanistic aspects of IPTG (isopropylthio-ß-galactoside) transport across the cytoplasmic membrane of Escherichia coli-a rate limiting step in the induction of recombinant protein expression.

Coupling transcription of a cloned gene to the lac operon with induction by isopropylthio-ß-galactoside (IPTG) has been a favoured approach for recombinant protein expression using Escherichia coli as a heterologous host for more than six decades. Despite a wealth of experimental data gleaned over this period, a quantitative relationship between extracellular IPTG concentration and consequent levels of recombinant protein expression remains surprisingly elusive across a broad spectrum of experimental conditions. This is because gene expression under lac operon regulation is tightly correlated with intracellular IPTG concentration due to allosteric regulation of the lac repressor protein (lacY). An in-silico mathematical model established that uptake of IPTG across the cytoplasmic membrane of E. coli by simple diffusion was negligible. Conversely, lacY mediated active transport was a rapid process, taking only some seconds for internal and external IPTG concentrations to equalize. Optimizing kcat and KM parameters by targeted mutation of the galactoside binding site in lacY could be a future strategy to improve the performance of recombinant protein expression. For example, if kcat were reduced whilst KM was increased, active transport of IPTG across the cytoplasmic membrane would be reduced, thereby lessening the metabolic burden on the cell and expediating accumulation of recombinant protein. The computational model described herein is made freely available and is amenable to optimize recombinant protein expression in other heterologous hosts. ONE-SENTENCE SUMMARY: A computational model made freely available to optimize recombinant protein expression in Escherichia coli other heterologous hosts.

A common inducer molecule enhances sugar utilization by Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1 is an electroactive bacterium that is a promising host for bioelectrochemical technologies, which makes it a common target for genetic engineering, including gene deletions and expression of heterologous pathways. Expression of heterologous genes and gene knockdown via CRISPRi in S. oneidensis are both frequently induced by ß-D-1-thiogalactopyranoside (IPTG), a commonly used inducer molecule across many model organisms. Here, we report and characterize an unexpected phenotype; IPTG enhances the growth of wild-type S. oneidensis MR-1 on the sugar substrate N-acetylglucosamine (NAG). IPTG improves the carrying capacity of S. oneidensis growing on NAG while the growth rate remains similar to cultures without the inducer. Extracellular acetate accumulates faster and to a higher concentration in cultures without IPTG than those with it. IPTG appears to improve acetate metabolism, which combats the negative effect that acetate accumulation has on the growth of S. oneidensis with NAG. We recommend using extensive experimental controls and careful data interpretation when using both NAG and IPTG in S. oneidensis cultures.

Soluble Expression of Antimicrobial Peptide BSN-37 from Escherichia coli by SUMO Fusion Technology.

Antimicrobial peptides (AMPs) are a kind of small molecular peptide that an organism produces to resist the invasion of foreign microorganisms. AMP BSN-37 is a bovine AMP that exhibits high antibacterial activity. In this paper, the optimized gene AMP BSN-37 was cloned into pCold-SUMO for fusion expression by recombinant DNA technology. The gene sequence of AMP BSN-37 was obtained by codons reverse translation, and the codons were optimized according to the codons preference of Escherichia coli (E. coli). The recombinant plasmid was constructed and identified by PCR, enzyme digestion and sequencing. Then the recombinant plasmid was transformed into BL21 E. coli to induce expression, and the IPTG concentration and time were optimized. The expressed soluble fusion protein SUMO-BSN-37 was purified by chromatography and then cleaved by SUMO proteases to release BSN-37. SDS-PAGE electrophoresis and Western blotting were used for identification. The recombinant plasmid pCold-SUMO-BSN-37 was obtained, and the fusion AMP BSN-37 was preliminarily expressed in BL21. After optimization, the optimal expression condition was 37 ℃ with 0.4 µM IPTG and 6 h incubation. Under optimal conditions, a large amount of fusion AMP BSN-37 was obtained by purification. Western blotting showed that the fusion peptide was successfully expressed and had good activity. The expressed BSN-37 showed antimicrobial activity similar to that of synthesized BSN-37. In this study, soluble expression products of AMP BSN-37 were obtained, and the problem regarding the limited source of AMP BSN-37 could be effectively solved, laying a foundation for further research on AMP BSN-37.

IPTG-induced high protein expression for whole-cell biosynthesis of L-phosphinothricin.

BACKGROUND: Biocatalytic production of L-phosphinothricin (L-PPT) is currently the most promising method. In this work, we use an Escherichia coli strain coexpressing of D-amino acid oxidase and catalase (E. coli DAAO-CAT) to oxidation biocatalytic D-PPT to PPO, then use the second E. coli strain coexpressing glutamate dehydrogenase and formate dehydrogenase (E. coli GluDH-FDH) to reduce biocatalytic PPO to L-PPT. MAIN METHODS AND MAJOR RESULTS: We compared the effects of different concentrations of IPTG or lactose on protein expression and enzyme activity in 5 L fermenter. The best induction conditions for E. coli DAAO-CAT were 0.05 mM IPTG, induction for 18 h at 28°C. The specific enzyme activities of DAAO and CAT were 153.20 U g-1 and 896.23 U g-1 , respectively. The optimal induction conditions for E. coli GluDH-FDH were 0.2 mM IPTG, induction for 19 h at 28°C. The specific enzyme activities of GluDH and FDH were 41.72 U g-1 and 109.70 U g-1 , respectively. The 200 mM D-PPT was biocatalyzed by E. coli DAAO-CAT for 4 h with space-time yield of 9.0 g·L-1 ·h-1 and conversion rate of over 99.0%. Then 220 mM PPO was converted to L-PPT by E. coli GluDH-FDH for 3 h with space-time yield of 14.5 g·L-1 ·h-1 and conversion rate of over 99.0%. To our knowledge, this is the most efficient biocatalytic reaction for L-PPT production. CONCLUSIONS AND IMPLICATIONS: We found that IPTG has advantages compared with lactose in the enzyme activity and biomass of E. coli DAAO-CAT and E. coli GluDH-FDH, and IPTG is more environmentally friendly. Our data implicated that IPTG can replace lactose in terms of economic feasibility and effectiveness for scaled-up industrial fermentations.

Enhanced Biomass Production of Recombinant Pfu DNA Polymerase Producer Escherichia coli BL21(DE3) by Optimization of Induction Variables Using Response Surface Methodology.

Pfu DNA polymerase is one of the most preferred molecular enzymes that is isolated from the hyperthermophilic Pyrococcus furiosus and used for high-throughput DNA synthesis by the polymerase chain reaction. Therefore, an efficient Pfu DNA polymerase production method is necessary for molecular techniques. In the present study, Pfu DNA polymerase was expressed in recombinant Escherichia coli BL21(DE3) and significant parameters for the biomass production were optimized using the central composite design which is the most popular method of response surface methodology. Induction conditions including cell density prior induction (OD600nm), post-induction temperature, IPTG concentration, and post-induction time and their interactions on biomass production were investigated. The maximum biomass production (14.1 g/L) in shake flasks was achieved using the following predicted optimal conditions: OD600nm before induction of 0.4 and the induction at 32 °C for 7.7 h, with 0.6 mM IPTG. Optimized culture conditions were implemented to scale up experiments. 22% and 70% increase in biomass production was achieved in 3 L and 10 L bioreactors, respectively as compared to initial biomass production observed in unoptimized conditions. Similary, a 30% increase of Pfu DNA polymerase production was obtained after the optimization. The polymerase activity of the purifed Pfu DNA polymerase was assessed by PCR amplification and determined as 2.9 U/µl by comparison with commercial Pfu DNA polymerase. The findings of this study indicated that the proposed fermentation conditions will contribute to further scale­up studies to enhance the biomass for the production of other recombinant proteins.

Potent IPTG-inducible integrative expression vectors for production of recombinant proteins in Bacillus subtilis.

The IPTG-inducible promoter family, Pgrac, allows high protein expression levels in an inducible manner. In this study, we constructed IPTG-inducible expression vectors containing strong Pgrac promoters that allow integration of the transgene at either the amyE or lacA locus or both loci in Bacillus subtilis. Our novel integrative expression vectors based on Pgrac promoters could control the repression of protein production in the absence and the induction in the presence of an inducer, IPTG. The ß-galactosidase (BgaB) protein levels were 9.0%, 15% and 30% of the total cellular protein in the B. subtilis strains carrying single cassettes with the Pgrac01, Pgrac100 or Pgrac212 promoters, respectively. The maximal induction ratio of Pgrac01-bgaB was 35.5 while that of Pgrac100-bgaB was 7.5 and that of Pgrac212-bgaB was 9. The inducible expression of GFP and BgaB protein was stably maintained for 24 h, with the highest yield of GFP being 24% of cell total protein while the maximum amount of BgaB was found to be 38%. A dual integration of two copies of the gfp+ gene into the B. subtilis genome at the lacA and amyE loci resulted in a yield of about 40% of total cellular protein and a 1.74-fold increase in GFP compared with single-integrated strains containing the same Pgrac212 promoter. The capability of protein production from low to high levels of these inducible integrative systems is useful for fundamental and applied research in B. subtilis.

Construction of a constitutively active type III secretion system for heterologous protein secretion.

Proteins comprise a multibillion-dollar industry in enzymes and therapeutics, but bacterial protein production can be costly and inefficient. Proteins of interest (POIs) must be extracted from lysed cells and inclusion bodies, purified, and resolubilized, which adds significant time and cost to the protein-manufacturing process. The Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS) has been engineered to address these problems by secreting soluble, active proteins directly into the culture media, reducing the number of purification steps. However, the current best practices method of T3SS pathway activation is not ideal for industrial scaleup. Previously, the T3SS was activated by plasmid-based overexpression of the T3SS transcriptional regulator, hilA, which requires the addition of a small molecule inducer (IPTG) to the culture media. IPTG adds significant cost to production and plasmid-based expression is subject to instability in large-scale fermentation. Here, we modulate the upstream transcriptional regulator, hilD, to activate the T3SS via three distinct methods. In doing so, we develop a toolbox of T3SS activation methods and construct constitutively active T3SS strains capable of secreting a range of heterologous proteins at titers comparable to plasmid-based hilA overexpression. We also explore how each activation method in our toolbox impacts the SPI-1 regulatory cascade and discover an epistatic relationship between T3SS regulators, hilE and the hilD 3' untranslated region (hilD 3'UTR). Together, these findings further our goal of making an industrially competitive protein production strain that reduces the challenges associated with plasmid induction and maintenance. KEY POINTS: • Characterized 3 new type III secretion system (T3SS) activation methods for heterologous protein secretion, including 2 constitutive activation methods. • Eliminated the need for a second plasmid and a small molecule inducer to activate the system, making it more suitable for industrial production. • Discovered new regulatory insights into the SPI-1 T3SS, including an epistatic relationship between regulators hilE and the hilD 3' untranslated region.

Enhancing the expression of multi-antigen chimeric TGAGS/BST protein from Toxoplasma gondii in Escherichia coli BL 21 Star during batch cultivation.

Toxoplasmosis, despite advances in science and technology, is a disease that requires attention since there is no vaccine capable of immunizing humans and animals against all isolated types of Toxoplasma gondii. Thus, the use of chimeric proteins, which can contain multiple antigens, is a very promising alternative for the process of obtaining a vaccine and diagnostic test for toxoplasmosis due to the great diversity of antigens presented by T. gondii. In this context, the present study evaluates batch culture strategies in the production of the multi-antigenic chimeric protein TgAGS/BsT from Toxoplasma gondii. Several exploratory cultures were initially carried out to observe the kinetic behavior of E. coli BL21 Star in five different medium compositions without the addition of IPTG (inducer). Cultures of E. coli B21 Star were carried out with 1.0 mM IPTG at different times of initiation of induction (0.5, 1, and 6 h) to evaluate the effects on cell growth, production of the protein of interest, culture pH, and acetic acid formation. The results showed that among the culture media evaluated, 2xTY and TB supplemented with glycerol had the best cell concentration values of 3.42 ± 0.05 g/L and 5.48 ± 0.05 g/L, respectively. In the assays induced by IPTG, a higher expression of TgAGS/BsT protein was observed, with induction beginning within 6 h of culture, with a maximum concentration of protein of interest of 1.82 ± 0.02 g/L for the 2xTY and 2.49 ± 0.03 g/L for the TB medium. In addition, later induction by IPTG provided greater stability of plasmid pET-TgAGS, remaining with values above 90% at the end of culture.

Cloning, Expression, and Purification of the Human Synthetic Survivin Protein in Escherichia coli Using Response Surface Methodology (RSM).

Survivin is one of the novel members of the apoptosis inhibitor protein family in humans. The main activity of the Survivin protein is to suppress caspases activity resulting in negative regulation of apoptosis. Survivin protein can be a potential target for the treatment of cancers between cancerous and normal cells. In the present research, the synthetic Survivin gene with PelB secretion signal peptide was cloned into a prokaryotic expression vector pET21a. The recombinant plasmid pET21a-PelB-Surv was expressed in Escherichia coli (E.coli) BL21, and the relative molecular mass of expressed protein was calculated 34,000 g/mol, approximately. The recombinant protein was purified through chromatography column and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Response surface methodology (RSM) was used to design 20 experiments for optimization of IPTG concentration, post-induction period, and cell density of induction (OD600). The optimum levels of the selected parameters were successfully determined to be 0.28 mM for IPTG concentration, 10 h for post-induction period, and 3.40768 for cell density (OD600). These findings resulted in 4.14-fold increases in the Survivin production rate of optimum expression conditions (93.6363 mg/ml).

Application of an Autoinduction Strategy to Optimize the Heterologous Production of an Antitumor Bispecific Fusion Protein Based on the TRAIL Receptor-Selective Mutant Variant in Escherichia coli.

Autoinduction is a simple approach for heterologous protein expression that helps to achieve the high-level production of recombinant proteins in soluble form. In this work, we investigated if the application of an autoinduction strategy could help to optimize the production of bifunctional protein SRH-DR5-B, the DR5-specific TRAIL variant DR5-B fused to a VEGFR2-specific peptide SRHTKQRHTALH for dual antitumor and antiangiogenic activity. The protein was expressed in Escherichia coli SHuffle B T7, BL21(DE3), and BL21(DE3)pLysS strains. By IPTG induction, the highest expression level was in SHuffle B T7, while by autoinduction, the similar expression level was achieved in BL21(DE3)pLysS. However, in SHuffle B T7, only 45% of IPTG-induced SRH-DR5-B was expressed in soluble form, in contrast to 75% autoinduced in BL21(DE3)pLysS. The yield of purified SRH-DR5-B protein expressed by autoinduction in BL21(DE3)pLysS was 28 ± 4.5 mg per 200 ml of cell culture, which was 1.4 times higher than the yield from IPTG-induced SHuffle B T7. Regardless of the production method, SRH-DR5-B was equally cytotoxic to BxPC-3 human tumor cells expressing DR5 and VEGFR2 receptors. Thus, the production of SRH-DR5-B by autoinduction in the E. coli BL21(DE3)pLysS strain is an efficient, technologically simple, and economical technique that allows to obtain a large amount of active protein from the cytoplasmic cell fraction. Our work demonstrates that the strategy of induction of protein expression is no less important than the strain selection.

Cloning and expression of recombinant human superoxide dismutase 1 (hSOD1) in Bacillus subtilis 1012.

OBJECTIVE: We aimed to clone and express the human Cu, Zn superoxide dismutase (hSOD1) in Bacillus subtilis 1012. Also, we investigated the expression level of hSOD1 under different induction conditions. RESULT: As an essential member of the antioxidant defense system in vivo, hSOD1 has become a therapeutic agent against host diseases, such as oxygen toxicity, acute inflammation, and radiation injury. The recombinant hSOD1 was successfully secreted extracellularly into B. subtilis 1012. The expression conditions were optimized, including inoculum size, different media, temperatures, and inducer concentrations. Finally, the highest level of hSOD1 was produced as a soluble form in Super rich medium by 2% inoculum with 0.2 mM of IPTG at 37 °C after the induction for 24 h. Besides, 20 g/L of lactose also displayed the same inductive effect on hSOD1 expression as that of IPTG (0.2 mM). Finally, the specific activity of purified hSOD1 was determined to be 1625 U/mg in the presence of 800 µM of Cu2+ and 20 µM of Zn2+. CONCLUSIONS: We propose that the B. subtilis 1012-hSOD1 strain system has great potential in future industrial applications.

[Preparation and activity evaluation of insulin-like growth factor 1 based on protein structure prediction].

In order to prepare insulin-like growth factor 1 (IGF-1) more economically and efficiently, the structure prediction and molecular docking of three IGF-1 fusion proteins were performed by computer simulation. The most suitable expression form of IGF-1 fusion protein was screened out. A prokaryotic expression vector of IGF-1 fusion protein was constructed and transformed into Escherichia coli Origami B(DE3) strain to obtain the recombinant strain. After induction with IPTG, the target protein was purified from the soluble fractions of the bacteria cell lysate by affinity chromatography, desalination, thrombin digestion and affinity chromatography of the enzyme digested products. An activity evaluation system was established by 3T3 cell proliferation method and the activity of the obtained IGF-1 was measured. The results showed that the sequence of the IGF-1 fusion protein prokaryotic expression vector was correct and the fusion protein was soluble upon 0.05 mmol/L IPTG induction at 25 ℃ for 16 h. After preliminary purification, thrombin digestion and re-purification, IGF-1 target protein with purity over 90% was obtained. Using the established activity evaluation system, the specific activity of IGF-1 was 2.47×105 U/mg, which was close to the standard product available at the market. The preparation technology of IGF-1 developed in this study may facilitate the development and industrial production of IGF-1 drugs.

Cloning, expression and seroreactivity of the recombinant lipopolysaccharide assembly protein - D (LptD) from Bartonella bacilliformis. / Clonamiento, expresión y seroreactividad de la proteína recombinante de ensamblaje de lipopolisacáridos - D (LptD) de Bartonella bacilliformis.

OBJECTIVE.: To evaluate in silico and at the serological level the antigenic potential of the recombinant extracellular domain of the lipopolysaccharide assembly protein - D (LptD) of Bartonella bacilliformis (dexr_LptD). MATERIALS AND METHODS.: Through in silico analysis, we selected a B. bacilliformis protein with antigenic and immunogenic potential. The selected protein gene was cloned into Escherichia coli TOP10 and expressed in Escherichia coli BL21 (DE3) pLysS. Recombinant protein was expressed using isopropyl-ß-D-1-thiogalactopyranoside (IPTG) and induction conditions were optimized. Finally, it was purified with Ni-IDA resin (His60 Ni Superflow) and a Western Blot assay was conducted. RESULTS.: In silico, the selected protein was LptD because it is located in the outer membrane and is antigenic and immunogenic. Optimized conditions for dexr_LptD induction were 0.5 mM IPTG, 16 hours, TB (Terrific Broth) medium, 3% (v/v) ethanol, 28 ºC, OD600: 1-1.5 and 200 rpm. Purification was carried out under denaturating conditions on a small scale and we obtained 2.6 µg/mL of partially purified dexr_LptD. The Western Blot assay showed a positive reaction between the sera from patients with Carrión's Disease and dexr_LptD, which shows the antigenicity of dexr_LptD. CONCLUSIONS.: The dexr_LptD shows antigenicity both in silico and at the serological level, these results are the basis for further studies on vaccine candidates against Carrion's Disease.

OBJETIVO.: Evaluar in silico y a nivel serológico el potencial antigénico del dominio extracelular recombinante de la proteína de ensamblaje de lipopolisacáridos - D (LptD) de Bartonella bacilliformis (dexr_LptD). MATERIALES Y MÉTODOS.: Mediante el análisis in silico se realizó la selección de una proteína de B. bacilliformis con potencial antigénico e inmunogénico. El gen de la proteína seleccionada se clonó en Escherichia coli TOP10 y se expresó en Escherichia coli BL21 (DE3) pLysS. La proteína recombinante fue expresada usando isopropil-ß-D-1-tiogalactopiranósido (IPTG) y se optimizaron las condiciones de inducción. Por último, se purificó con resina Ni-IDA (His60 Ni Superflow) y se realizó un ensayo de Western Blot. RESULTADOS.: In silico, la proteína seleccionada fue LptD por estar localizada en la membrana externa y ser antigénica e inmunogénica. Las condiciones optimizadas para la inducción del dexr_LptD fueron 0,5 mM IPTG, 16 h, medio TB (Terrific Broth), etanol al 3% (v/v), 28 ºC, OD600: 1-1,5 y 200 r.p.m. La purificación se realizó en condiciones denaturantes a pequeña escala y se obtuvo 2,6 µg/mL de dexr_LptD parcialmente purificada. El ensayo de Western Blot mostró una reacción positiva entre los sueros provenientes de pacientes con la enfermedad de Carrión y dexr_LptD, ello evidencia la antigenicidad del dexr_LptD. CONCLUSIONES.: El dexr_LptD muestra antigenicidad in silico y a nivel serológico, estos resultados son base para posteriores estudios sobre candidatos vacunales contra la enfermedad de Carrión.

Development and Application of Two Inducible Expression Systems for Streptococcus suis.

Streptococcus suis is an important zoonotic bacterial pathogen posing a threat to the pig industry as well as public health, for which the mechanisms of growth and cell division remain largely unknown. Developing convenient genetic tools that can achieve strictly controlled gene expression is of great value for investigating these fundamental physiological processes of S. suis. In this study, we first identified three strong constitutive promoters, Pg, Pt, and Pe, in S. suis. Promoter Pg was used to drive the expression of repressor genes tetR and lacI, and the operator sequences were added within promoters Pt and Pe. By optimizing the insertion sites of the operator sequence, we successfully constructed an anhydrotetracycline (ATc)-inducible expression system and an isopropyl-ß-d-thiogalactopyranoside (IPTG)-inducible expression system in S. suis. We showed that these two systems provided inducer-concentration- and induction-time-dependent expression of the reporter gene. By using these tools, we investigated the subcellular localization of a key cell division protein, FtsZ, which showed that it could be correctly localized to the midcell region. In addition, we constructed a conditional knockout strain for the glmS gene, which is an essential gene, and showed that our ATc-inducible promoter could provide strictly controlled expression of glmS in trans, suggesting that our inducible expression systems can be used for deletion of essential genes in S. suis. Therefore, for the first time we developed two inducible expression systems in S. suis and showed their applications in the study of an important cell division protein and an essential gene. These genetic tools will further facilitate the functional study of other important genes of S. suis. IMPORTANCE Streptococcus suis is an important zoonotic bacterial pathogen. Studying the mechanisms of cell growth and division is important for the identification of novel antimicrobial drug targets. Inducible expression systems can provide strictly controlled expression of the protein of interest and are useful tools to study the functions of physiologically important proteins. However, there is a lack of convenient genetic tools that can achieve inducible protein expression in S. suis. In this study, we developed two (ATc-inducible and IPTG-inducible) inducible expression systems and showed their applications in a subcellular localization study of a cell division protein and the construction of conditional knockout of essential genes in S. suis. These systems will be useful for functional studies of important proteins of S. suis.

Optimization of an Inclusion Body-Based Production of the Influenza Virus Neuraminidase in Escherichia coli.

Neuraminidase (NA), as an important protein of influenza virus, represents a promising target for the development of new antiviral agents for the treatment and prevention of influenza A and B. Bacterial host strain Escherichia coli BL21 (DE3)pLysS containing the NA gene of the H1N1 influenza virus produced this overexpressed enzyme in the insoluble fraction of cells in the form of inclusion bodies. The aim of this work was to investigate the effect of independent variables (propagation time, isopropyl ß-d-1-thiogalactopyranoside (IPTG) concentration and expression time) on NA accumulation in inclusion bodies and to optimize these conditions by response surface methodology (RSM). The maximum yield of NA (112.97 ± 2.82 U/g) was achieved under optimal conditions, namely, a propagation time of 7.72 h, IPTG concentration of 1.82 mM and gene expression time of 7.35 h. This study demonstrated that bacterially expressed NA was enzymatically active.

(Optochemical) Control of Synthetic Microbial Coculture Interactions on a Microcolony Level.

Synthetic microbial cocultures carry enormous potential for applied biotechnology and are increasingly the subject of fundamental research. So far, most cocultures have been designed and characterized based on bulk cultivations without considering the potentially highly heterogeneous and diverse single-cell behavior. However, an in-depth understanding of cocultures including their interacting single cells is indispensable for the development of novel cultivation approaches and control of cocultures. We present the development, validation, and experimental characterization of an optochemically controllable bacterial coculture on a microcolony level consisting of two Corynebacterium glutamicum strains. Our coculture combines an l-lysine auxotrophic strain together with a l-lysine-producing variant carrying the genetically IPTG-mediated induction of l-lysine production. We implemented two control approaches utilizing IPTG as inducer molecule. First, unmodified IPTG was supplemented to the culture enabling a medium-based control of the production of l-lysine, which serves as the main interacting component. Second, optochemical control was successfully performed by utilizing photocaged IPTG activated by appropriate illumination. Both control strategies were validated studying cellular growth on a microcolony level. The novel microfluidic single-cell cultivation strategies applied in this work can serve as a blueprint to validate cellular control strategies of synthetic mono- and cocultures with single-cell resolution at defined environmental conditions.

Alternative Heterologous Expression of L-Arabinose Isomerase from Enterococcus faecium DBFIQ E36 By Residual Whey Lactose Induction.

This study reports an alternative strategy for the expression of a recombinant L-AI from Enterococcus faecium DBFIQ E36 by auto-induction using glucose and glycerol as carbon sources and residual whey lactose as inducer agent. Commercial lactose and isopropyl ß-D-1-thiogalactopyranoside (IPTG) were also evaluated as inducers for comparison of enzyme expression levels. The enzymatic extracts were purified by affinity chromatography, characterized, and applied in the bioconversion of D-galactose into D-tagatose. L-AI presented a catalytic activity of 1.67 ± 0.14, 1.52 ± 0.01, and 0.7 ± 0.04 U/mL, when expressed using commercial lactose, lactose from whey, and IPTG, respectively. Higher activities could be obtained by changing the protocol of enzyme extraction and, for instance, the enzymatic extract produced with whey presented a catalytic activity of 3.8 U/mL. The specific activity of the enzyme extracts produced using lactose (commercial or residual whey) after enzyme purification was also higher when compared to the enzyme expressed with IPTG. Best results were achieved when enzyme expression was conducted using 4 g/L of residual whey lactose for 11 h. These results proved the efficacy of an alternative and economic protocol for the effective expression of a recombinant L-AI aiming its high-scale production.

In Vivo Feedback Control of an Antithetic Molecular-Titration Motif in Escherichia coli Using Microfluidics.

We study both in silico and in vivo the real-time feedback control of a molecular titration motif that has been earmarked as a fundamental component of antithetic and multicellular feedback control schemes in E. coli. We show that an external feedback control strategy can successfully regulate the average fluorescence output of a bacterial cell population to a desired constant level in real-time. We also provide in silico evidence that the same strategy can be used to track a time-varying reference signal where the set-point is switched to a different value halfway through the experiment. We use the experimental data to refine and parametrize an in silico model of the motif that can be used as an error computation module in future embedded or multicellular control experiments.