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GENE EXPRESSION: WHICH PLASMID (Lux or pUC18) IS MORE EFFECTIVE FOR E. coli TRANSFORMATION?

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Abstract

The role of plasmids in bacterial transformation with subsequent use in gene cloning and expression studies cannot be overemphasized. The concept of this work was to explicitly determine the effect of transformation of two different plasmids lux and pUC18 on the cellular phenotypic expression. Preparation of competent cells was first carried out using the Calcium Chloride technique and then transformation was carried out using desired plasmids lux and pUC18. Agar based ampicillin treatments were adopted, and the colonies formed were subsequently counted, after incubation. To control the experiments, cells without plasmid treatments were also observed. At the end of the experiments, it was observed that the treatments of plates without ampicillin showed luxuriant lawn growths while the plates carrying ampicillin had a less luxuriant growth of E. coli in the form of distinct colonies. The distinct colonies were counted and the treatment of lux plasmid had 17 colonies while the control treatment of pUC18 had 5 colonies. The treatments without plasmids were also showing corresponding no growth and lawn growth observations when plated out on ampicillin and non-ampicillin containing media respectively. The experiments showed that the lux plasmid was a more efficient plasmid than the pUC18, and it was conclusively opined that the effect of quorum sensing mechanism of the lux operon led to the higher efficiency recorded.

Introduction

Transformation in bacteria is the term used to explain the natural uptake of external DNA from the immediate surrounding or environment of the bacteria. With the acquisition of the plasmid DNA the encoded phenotypic traits of the plasmid DNA are then conferred on the host cell, leading to some benefit for the host. A good example of such trait is antibiotic resistance. However, the host organism can survive effectively in natural environments with or without the presence of any external plasmid, thus plasmids could be said to be exotic extrachromosomal DNA that have ability to transform natural non-harboring bacterial cells to carrier cells thereby influencing their metabolism. The opening of new research studies in Molecular cloning since its advent in 1970, has had a basic target of artificially applying recombinant plasmid DNA into competent cells of Escherichia coli as a model organism for these studies (Rosano and .Ceccarelli 2014). This has broadened the horizon of the science of cloning which essentially starts by transformation of the organism using a desired vector (either a plasmid or bacteriophage).

The targeted use of plasmids to artificially transform E. coli has been a major subject of study over time. Hanahan (1983) stated that a couple of factors were responsible for the complete genetic transformation of E. coli cells and attempted to investigate some of those factors. In his statements, competency of the host bacterial cell was key. As earlier stated, the cells used in such study have to be made ‘competent’. Competency is basically the process of making the cells more receptive to DNA uptake by virtue of increased cellular permeability. Hanahan (1983) focused the research on the improvement of methods of preparing competent cells with optimized nutritional requirements with the aim of achieving higher transformation efficiency. For a successful transformation, there are certain considerations which should be taken. There is a need to determine the host cell/strain into which the extrachromosomal DNA can possibly be introduced into. Subsequently it is pertinent to fully determine the delivery method to be adopted in inserting the DNA into the host, as well as a means of identification and selection of successfully transformed cells. The Even with this there seem to be other major factors that affect the efficiency of a plasmid as it transforms E.coli cells, and Liu et al. (2014) have stated that some of those factors are plasmid specific so long as the expression host remains te same strain of E.coli.

The inference of the use of antibiotic resistance coding plasmids in transforming E.coli cells should yield phenotypic expressions of resistance within cells which were hitherto susceptible to the test antibiotic. The ability to detect and quantify the level of resistance acquired is best described experimentally by using specific antibiotic resistance coding plasmids to determine their efficacy in terms of colony counts on nutrient plates. Such counts are essential in qualifying the efficiency level of the plasmid and shed more light on the molecular biology of the plasmid as well as the transformation process.

Experimentally, plasmids can be inserted into the host cells via chemical and physical methods. The chemical method which is the core focus of this write-up centers around the use of Calcium Chloride to induce competency in E.coli. Earlier publications by Mandel and Higa (1970) explained that there was an increased level of permeability of the cells after they had been exposed to Calcium ions (Ca2+) at elevated temperatures. Their work preceded the fine-tuning as carried out by Hanahan (1983) on the process. Still, there are some plasmid-specific variations that occur with respect to transformations.

This experimental procedure in this work was targeted at observing the efficiency of two plasmids: (a) pUC18 plasmid –with molecular weight of 2×106, carrying a penicillin/ampicillin-resistance gene that has the possibility of conferring antibiotic resistance on host E. coli cells, with untransformed cells lacking the ability to proliferate in ampicillin rich medium. and (b) lux plasmid – with molecular 4.5×106, bearing the  lux operon obtained from the bioluminescent bacteria Vibrio fischeri which emits light and gives a glowing phenotypic appearance. The independent expressions of these two genes were assessed in this work, and observation made.

METHODS

The experimental methods adopted involved the use of plasmid lux as the test plasmid and the plasmid pUC18 as the control. The two plasmids were transformed into E.coli adopting the chemical transformation method of competent cells with the aid of Calcium chloride. For competent cell preparation, a vial of CaCl2 solution was placed in an ice bath to yield cold CaCl2. Following aseptic techniques, 590 uL of cold CaCl2 was transferred into a tube containing 50 uL of E. coli. The tube was mixed thoroughly and ten incubated on ice for 10 minutes. The cells within this system were regarded as competent. Plasmid uptake by competent cells were subsequently demonstrated by using 3 uL each of test plasmid (lux) and control plasmid (pUC18) in tubes labeled accordingly (and kept cold in ice). Using a pipette, 70 uL of earlier prepared competent cells were transferred into each of the tubes containing the plasmids. Te plasmid-cell mixtures were stored on ice for 15 minutes. Similar volume of competent cells without plasmids were also placed in tubes (on ice) and labeled accordingly. All tubes were then subsequently transferred into a water bath of pre-set temperature of 37oC and allowed to stand for 5 minutes. About 300 uL of nutrient broth (NB) was subsequently introduced into the tubes containing plasmids and 150 uL NB into the non-plasmid containing tubes. All tubes were incubated at 37oC for 45 minutes.

Agar plate technique was then used to determine the efficiency of the transformation on ampicillin and non-ampicillin containing plates. One hundred and thirty micro liters of mixed bacterial suspension from the tube with the ‘control’ plasmid was obtained using a sterile pipette and aseptically dispensed unto the surface of the ampicillin-containing agar plates. A sterile glass spreader was then used to disperse the cells evenly on the surface. Similarly, the tubes containing bacterial suspension of ‘test’ plasmid were also plated out on agar medium. The tubes with ‘no plasmids’ were also plated out. The plates were allowed to stand at room temperature for 10 minutes before incubating at 37oC. Similar procedure was carried out for non-ampicillin containing plates.

After incubation, the plates were obtained and viewed for lawn or colonial growth of E.coli. The plates were also viewed in the dark and observations were recorded.

RESULTS

Table 1 shows the growth observations (phenotypic expressions) on the plates after the experiments. Plates without ampicillin showed luxuriant lawn growths while the plate carrying ampicillin had a less luxuriant growth of E. coli in the form of distinct colonies. The treatments without plasmids were also showing corresponding no growth and lawn growth observations when plated out on ampicillin and non-ampicillin containing media respectively. Figure 1 corroboratively shows the pictures of plates with colonies after incubation

Table 1: Plasmid/Cell treatments of lux and pUC18 plasmids and their phenotypic expressions on agar plates

Treatment

Observation

Colony count for colonial growth

LBlux

Lawn growth

NA

LBc

Lawn growth

NA

LB/AMPlux

Colonial growth

17

LB/AMPc

Colonial growth

5

LBnp

Lawn growth

NA

LB/AMPnp

No growth

0

 

Key:

LBlux – lux plasmid treatment grown on media without ampicillin

LBc – control plasmid (pUC18) treatment grown on media without ampicillin

LB/AMPlux – lux plasmid treatment grown on ampicillin containing medium

LB/AMPc – control plasmid treatment grown on ampicillin containing medium

LBnp – no plasmid treatment grown on media without ampicillin

LB/AMPnp – no plasmid treatment grown on media with ampicillin.

NA – Not applicable

 

Figure 1: Picture of plates with E. coli/plasmid test treatments and control

DISCUSSION

Plasmid based transformations of bacterial cells are a very unique tool in current molecular biology studies. The application of these two distinct plasmids (lux and pUC18) in the same E.coli strain was the focus of this work. The concept was to determine cell transformation and at the same time determine which of the plasmids had a better efficiency level in terms of host cell transformation. From results of this experiment,  transformation via the Calcium chloride treatment was observed for all cells tested as expected. Clark et al. (2002) stated that E. coli cells interact with DNA more actively in the presence of divalent cations thus facilitating higher efficiency of transformation at the early stages of DNA uptake. This was as obtained when cold CaCl2 was applied. Madison and Huisman (1999) also explained that poly-3-hydroxybutyrates which are low molecular weight compounds produced within the cell have been known to form a complex wit Ca2+ channels within the bacterial membrane, as they facilitate DNA import after Calcium treatment, thus leading to the competent natures of the cells. The effect of calcium chloride as an agent of conferring competence on E. coli cells has been proven in various experiments previously (Liu et al., 2014) as well as this current experiment. 

In line with the experimental efficacy of the two plasmids tested, the results showed that the two plasmids behaved differently with the lux gene yielding a higher number of transformed colonies than the pUC18 control. This consequently means that the lux plasmid was a better plasmid for transformation than the pUC18. Properties of the two plasmids showed variable sizes, with the lux plasmid weighing slightly higher than the control plasmid. According to earlier works by Hanahan (1983), the smaller the plasmid the higher its transformation efficiency. However this was not the case with these two plasmids as the higher molecular weight lux plasmid yielded a better result in terms of colony numbers. A plausible explanation is te fact that the lux plasmid which is a product of V. fischeri is controlled by a population density regulatory mechanism known as quorum sensing. (Dunlap, 1999). The chemical signal, 3-oxohexanoyl L-homoserine lactone is usually secreted in increasing cell populations further triggering increased cell numbers as it is secreted. The increased secretion of the signal molecule leads to the activation of the transcription of the the luxICDABEG genes and resulting light production. Based on the molecular biology of the lux genes within the plasmid, Fuqua et al. (1994), Milton, (2006), Ulitzur (1998), Waters and Bassler (2005), have all stated that  a second autoinducing molecule known as, octanoyl L-homoserine lactone (C8- HSL), may also stimulate the transcription of the lux genes. The difference in cell number between the normal control pUC18 which carries an ampicillin resistance phenotype alone and the bioluminescence production coding plasmid shows the effect quorum sensing has on gene expression

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