Examination of Pseudomonas Fluorescence as a Recombinant Expression Host: Cloning, expression , and Chromatography (Biomedical Project)

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ABSTRACT:

In an effort to expand the pool of bacterium useful for biotechnology applications, Pseudomonas fluorescens, a common gram negative microbe, was examined for its ability to function in a recombinant setting. P. fluorescens is ubiquitous in nature and was initially identified as a soil bacterium found in dirt and is typically associated with plant material.

Past literature indicates that it shared characteristics common to Escherichia coli and Bacillus subtilis, including simple growth conditions and potential cloning vectors, providing motivation to look into both the upstream and downstream characteristics of this bacterium. First, it was demonstrated that P. fluorescens could be grown to acceptable cell densities in simple batch with cell weights on the order of 60 g/L in the absence of optimization.

Lysates of cells were subjected to DEAE ion exchange chromatography to identify the subproteome of soluble proteins which are retained by this resin to guide cellular modifications that reduce the amount and number of host cell proteins (HCPs) encountered during bioseparation.

Finally, cloning experiments with Green Fluorescent Protein and FC fragment of tetanus demonstrated both moderate- and large- recombinant DNA products may be obtained from this host.

BACKGROUND

Proteome:

The term proteome arose in 1995 and was defined as the comprehensive study of cellular proteins under specific conditions at a given time (Wasinger et al., 1995). The purpose is not only to know their interactions and/or modifications, but also to create a multi-dimension view including time.

Knowledge of a proteome requires immense information on molecular biology, biochemistry, and bioinformatics. Normally, cells are undergoing natural and acquired differentiations and are varied in their ages; therefore, they may make or degrade protein(s). For this reason a cell is able to produce an uncountable number of proteins, since the cell is dynamicin nature making it unlike the static behavior of the  genome  (Eisenberg, Marcotte, Xenarios, & Yeates, 2000).

The Four Levels of Protein Structure.

The Four Levels of Protein Structure.

Biotechnology and bioprocesses:

In 1919, a Hungarian engineer, Karl Ereky, proposed that any product obtained from raw substances with the help of living organisms as biotechnology (Ereky, 1919; Fári & Kralovánszky, 2006).

However, this definition preceded modern techniques neither developed nor applied in the early 1900s. Nowadays, this biological era has evolved enough to permit researchers updated information on, for example, the central dogma, genetic materials, gene mutation, gene transformation, gene isolation and sequencing, functional enzymes, and much other essential information.

Vaccines:

Vaccines are biochemical substances given to living organisms to stimulate their immune response against certain infectious diseases, mainly bacteria and viruses. There are several recombinant vaccines and different methods used to obtain vaccines characterized by their specificity, effectivity, purification, and safety.

Before modern bioprocesses had been developed, the production of a vaccine depended on inactivation of the infectious agent or its toxic products. Inactivation was carried out mostly by repeat culturing of the virulent microbe or by treating the toxins with chemicals that alter their activity. Live attenuated and inactivated vaccines are examples of traditional techniques.

Pseudomonads:

The genus of pseudomonas contains species with much functional diversity. However, all are ubiquitous and live mainly in water, soil, and plant surfaces (Hirano & Upper, 2000). The genus belongs to γ-proteobacteria which is gram negative rods, motile, aerobic and is able to produce cytochrome oxidase enzyme.

Cytochrome oxidase catalyzes the oxidation of cytochromec and reduces oxygen to form water (Parr et l. 1974). Production of cytochrome oxidase enzyme is the main feature that differ Pseudomonas spp from other gram negative enterobacteriaceae, especially when clinical laboratories are providing identification.

Complete Genome Sequence of P. Fluorescenspf-5

Complete Genome Sequence of P. Fluorescenspf-5

SCOPE OF THE RESEARCH

Literature suggests an investigation of the ability for P. fluorescens to produce recombinant proteins, eventually leading to widespread adoption of this bacterium as a recombinant host. While it has been suggested that this bacterium could compete with E. coli or other simple prokaryotes, laying the groundwork for this to occur has not happened to a significant extent. This study examined the cloning and expression of two proteins in order to demonstrate P. fluorescens has potential in this regard. Specifically,

  • A plasmid was constructed that employed IPTG induction for protein expression.
  •  The expression of two recombinant products (Green Fluorescent Protein, and FC) was demonstrated.
  • Information about host cell proteins which may be encountered during ioseparation
    was collected.

MATERIALS AND METHODS

Preparation of P. fluorescens:

Pseudomonas fluorescens  ATCC949 was purchased from the American Type Culture Collection (ATCC) in lyophilized powdered form. Cultures were cultivated using the most routinely used growth medium named Luria-Bertani (LB) broth and agar because LB medium contains simple nutrient elements enough to supply the bacteria with essential growth requirements such as peptone, yeast extract, and  sodium chloride (Bertani, 1951). Half milliliter of Luria-Bertani (LB) broth was added to the powder and gently mixed to dissolve completely.

Antibiotic Sensitivity Tests:

Resistance and sensitivity tests against different antibiotics were carried out sing Kirby-Bauer Disk Diffusion Susceptibility Test Protocol (Bauer et al. 1966). A loop filled with P. fluorescens was spread over LB agar. Paper disks were soaked with various concentrations and types of antibiotics and put over the surface of the agar. After 18 hours, a clear zone of growth inhibition surrounding an antibiotic disk was considered sensitive, whereas, growth around resistant.

RESULTS AND DISCUSSION

Selection of Tetracycline Marker:

P. fluorescens is abundant in nature as  microbial flora of soil water and plants. Due to competition  within the microbiota, P.fluorescens produces inhibitors and antibacterial substances that permit survival in its niche. Depending on this fact, the strain was examined for antibiotic sensitivity to determine which one is  effective against the tested strain for future cloning.

After 17 hrs / 26°C of  rehydrated lyophilized powder of P.fluorescens on LB agar, visible colonies were  grown. Single colonies were isolated and cultured on the same agar and different antibiotics were put on the surface of the agar and incubated overnight at 26°C. Inhibition growth zones were shown around tetracycline and kanamycin disks,  whereas, the bacteria exhibited some growth around ampicillin.

Confirming the Transform of P. fluorescens with PGFPuv;

P. fluorescens has natural resistance to ampicillin, therefore to established pGFPuv (which also carries the resistance gene), the concentration of antibiotic in the agar and broth was increased from 100 μg/ml to 150 μg/ml. Detection of plasmid transformed in E. coli was proven by grown colonies on AMP containing-LB agar; whereas, confirmed in P. fluorescens by fluoresce illumination of P. fluorescens colonies on agar and in broth when exposed to UV light.

 Illumination of Pgfp-containing P. Fluorescens in Lb Broth (Left) and a Lb Agar (Right) Under Ultraviolet Light.

Illumination of Pgfp-containing P. Fluorescens in Lb Broth (Left) and a Lb Agar (Right) Under Ultraviolet Light.

CONCLUSIONS

  • Pseudomonas fluorescens was successfully grown in a batch fermenter, demonstrating the bacterium cold be grown to sufficient optical density to be an attractive alternative to E. coli  as a recombinant host.  Although not examined in this study, one rationale to choose P. fluorescens over the E. coli is the fact that this soil bacterium may display less burden on downstream processing in terms of endotoxicity.
  • Plasmids were constructed by taking elements from a vector originally designed to examine promoter efficiency in P. fluorescens. An IPTG-inducible cassette was incorporated into pBBR1mcs-3 which was used to over express Green Fluorescent Protein as a model protein.  This portion of the study demonstrated recombinant protein production is possible with this host.
  • DEAE of soluble protein from wild type P. fluorescens identified the most abundant HCPs that would be encountered during bioseparation via ion exchange chromatography. The large number of essential proteins indicated that gene repression may be a better strategy than simple gene knockout.
  • Transformation of P. fluorescens with a plasmid bearing a ColE1/pUC origin and the gene for Fragment C (FC) of Clostridium tetani demonstrated the potential of using existing plasmids with this organism. To our knowledge, this is the first report of the use of this strain to produce FC of tetanus toxoid.

Source: University of Arkansas
Author: Ahmed K.Ali Elmasheiti

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