Abstract
Background: Taq polymerase is an important tool in gene modification and engineering. This enzyme was first isolated from Thermus aquaticus but now commercially produced in E.coli. The technology of producing Taq polymerase in E.coli cells is being continually improved.
Methods: Plasmid was purified from E.coli cells using mini-prep isolation method, followed by EcoRI digest. Taq gene fragment was visualised in electrophoresis and harvested using QiagenTM gel purification kit. Plasmid pETBlue-1 served as vector to transform taq gene into competent E.coli cells using traditional CaCl2/heat shock transformation procedure. Blue-white screen was used to identify recombinant. After overexpression and purification of Taq polymerase in E.coli, we used SDS-PAGE to analyse the enzyme protein quality, along with gel electrophoresis to determine enzyme activity in PCR reactions. Concentration and purity of DNA samples were all determined by Nanodrop spectrophotometer.
Results: The methods used in this lab practice resulted in overall satisfactory outcome of cloning, expression and purification of Taq polymerase in E.coli cells. Despite the poor purity of extracted taq gene and low transformation efficiency, Taq polymerase was successfully produced and purified from E.coli, showing high enzyme efficiency in PCR reaction.
Conclusion: Following the standard procedures, we successfully produced Taq DNA polymerase with sufficient activity to setup PCR reactions.
Keywords: Taq polymerase, plasmid purification, recombinant protein expression, protein purification, PCR
Background
Taq polymerase was first isolated from hot water springs bacteria Thermus aquaticus [1] and now has been commonly used in DNA amplification and sequencing protocols. On account of the high demand of this enzyme, native polymerase was commercially replaced by recombinant Taq polymerase [2]. However at the beginning of this industry, Taq polymerase yield was very low due to the poor expression of taq gene in Escherichia coli [2]. In 1994, an effective overexpression system was successfully constructed. By altering codons around the N-terminal region the enzyme production was enhanced more than ten-fold [3]. Simultaneously in the early 1990s, simplified and improved methods to isolate Taq polymerase produced in E.coli were also reported [4, 5] Through decades of studies ranging from vector type to induction conditions [6, 7], the technology of cloning, expression and purification of Taq polymerase in E.coli has been greatly improved. Nowadays, technologies in this area continue to develop.
Results and discussions
Extraction and purification of plasmid
Sample S and sample D were obtained through mini-prep isolation of plasmid pTaq1 fromE.coli culture prepared by students and demonstrator respectively. Sample S and D generated distinct concentration of pTaq1, which are respectively 19.3 ng/μl and 175.5 ng/μl (Fig.1a). Although D cutures produced significantly higher quantities of plasmid than S culture, both samples performed similar purity according to the 260/280 ratios. The 260/280 ratio of both samples were over 1.9 (Fig.1a), indicating that the plasmid samples can be considered free from organic contaminations but might contain RNA residue [7-9]. During the purification precedure, proteins and genomic DNA were neuralised and preciptated by the application of neutralisation buffer. Meanwhile, RNase in cell suspension buffer degrated RNA and reduced RNA residue but this action might have low efficiency and resulted in the relatively high value of 260/280. To improve the efficiency of RNA degration, RNase can be applied at a higher concentration or allowed longer reaction time.
Mini-prep isolation utilises alkaline extraction method, the main product of which is covalently closed circular (CCC) plasmid DNA due to its conformational stability under alkaline treatment [7, 10, 11]. However the 1kb DNA ladder consists of linear DNA fragments, which have lower migration rates than supercoiled plasmid DNA (CCC form) through agarose gel [10, 12, 13]. Therefore, The length of plasmid should be estimated greater than the corresponding value on the DNA ladder. According to the gel electrophoresis result, the length of purified plasmid DNA should be above 3000 bp as both samples had produced the brightest band at 3000bp level on the DNA ladder (Fig.1b). In sample D, a small amount of nucleic acids separated from supercoiled plasmid DNA (band3) was also observed (Fig.1b), which accorded with the diverse conformational forms of bacterial plasmid. Band1 indicated multimer forms of plasmid, which was formed by monomers interlocking together [7]. Multimer forms of plasmid have very slow migration rate because of their larger molecular size. We also discovered a band (band2) between multimers and supercoiled plasmid DNA (Fig.1b). This was likely to be the open circular (OC) form of plasmid, also known as nicked double-stranded DNA, which migrated slower than CCC plasmid DNA but faster than multimers [10, 13]. Another minor band (band4) also appeared in Fig.1b. This band represented degraded RNA or RNA residue with low molecular weight [10, 13]. In comparison, plasmid sample purified from culture S produced only a single band of 3500bp DNA fragments, with relatively low intensity (Fig.1b). It can be explained that low bacterial concentration in culture S gave rise to low plasmid concentraition along with low quatities of other components that were not sufficient to be seen in gel electrophosis.
Taq gene purification
Samples were treated with restriction enzyme EcoRI, whose two sites on the pTaq1 plasmid flank the taq gene. EcoRI digestion yielded two bands, and the one measuring around 1800bp contained the taq gene (Fig.2a). The digestion of plasmid can be considered completed, since there were only two bands observed. Although the DNA sample contained diverse plasmid forms, they all resulted in identical fragments after the EcoRI digestion. EcoRI digestion produced linear double-stranded DNA fragments, thus the 1kb marker has accurate estimation to the length of these fragments. The size of pTaq1 can be estimated by adding together the length of band1 and band2, which was around 5000bp. Nevertheless, undigested pTaq1 (Fig.1b and Fig.2a) migrated appreciably faster than 5000bp linear fragments in the marker, on account of the particular high migration rate of supercoiled plasmid through agarose gel. The control groups presented similar band distribution to the plasmid in Fig.1a, suggesting that adjuvants (restriction buff and water) for digest process did not contaminate or affect the samples.
EcoRI digest was followed by gel purification to harvest the taq gene. Sample S yielded 5.7 ng/μl DNA with a 260/280 ratio of 2.42 and sample D yielded 9.1 ng/μl DNA with a 260/280 ratio of 2.12 (Fig.2c). A 260/280 ratio greater than 2 could be considered an indicator of high RNA concentration [7-9]. We conjectured that poor purity of DNA samples was caused by the RNA residue from plasmid samples. And the impact of RNA contamination on 260/280 ratio became more obvious when DNA concentration was very low. Gel purification of EcoRI digest product in sample D generated a small amount of 2000bp DNA fragment encoding the Taq polymerase, while sample S did not produce any visible band (Fig.2b), most likely due to the particularly low concentration of the product.
Expression of Taq DNA polymerase
Each colony was developed from a single bacterium successfully transformed with the plasmid. Most of the bacteria harbour recombinant plasmid but some are transformed with empty vectors (Table.1). The low concentration of tag gene in both samples (Fig.2c) failed to meet the 1:3 molar ratio of vector to insert, which accounted for the incomplete plasmid recombination. By comparing the result of sample D and sample S, we found that higher concentration of the insert DNA contributed to a higher recombination frequency (Fig.2c and Table.1). Ligation of taq DNA fragment to vector plasmid produced a transformation efficiency of 5.17×104 cfu/μg in sample S and 6.97×104 cfu/μg in sample D, which were acceptable but relatively low [7]. In comparison, positive control transformed with supercoiled plasmid showed a higher transformation efficiency of 8.32×104 cfu/μg. According to previous researches [7, 14], transformation efficiency is compromised by the restriction cleavage of vector plasmid and religation of cleaved vector has little or no recovery of the reduced transformation efficiency. During the heat shock phase, the temperature of waterbath fluctuated to around 47°C, which might have killed some bacteria and led to the overall low efficiency. Some researches have reported that increasing plasmid concentration along with vigorous shaking could contribute to higher transformation efficiency [14].
The recombinant Taq polymerase was purified from induced E.coli (pTaq2) cells that contained the taq gene. Culture containing control plasmid without the taq gene (pControl) was also included. The expression of Taq DNA polymerase was demonstrated by comparing the SDS-PAGE of the proteins present in cell lysate of E.coli (pTaq2) to those present in cell lysate of E.coli (pControl) (Fig.3). Accordingly, we confirmed Taq polymerase expression inE.coli (pTaq2) and the molecular weight of this enzyme was around 92 kDa. However, we also observed a slight amount of Taq polymerase in the control samples, which was caused by sample contamination from next lane leakage, since we loaded the sample too early before running.
PCR
Subsequently, we examined purified Taq enzyme activity through PCR performance. PCR reactions were setup with commercial Taq polymerase or with Taq polymerase purified fromE.coli (pTaq2). Template DNA was successfully amplified using either Taq enzyme, showing an 1100 bp DNA fragment in 1% agarose gels. Moreover, two individual PCRs have produced very similar band distribution and intensity, suggesting that Taq polymerase purified in lab had nearly identical enzyme activity to commercial Taq polymerase (Fig.4). E.coli (pControl) cell lysate obtained through a parallel purification process was used to setup PCR reaction and no DNA amplification product was observed. Successful amplification of DNA template via PCR validated the feasibility of producing Taq polymerase in recombinant E.coli.
Conclusion
In conclusion, the technologies involved in this lab practical include mini-prep DNA isolation,EcoRI restriction digest, gel electrophoresis, gel purification, plasmid recombination, plasmid transformation, protein overexpression, protein extraction, SDS-PAGE analysis and PCR. Following the standard procedures of cloning and overexpressing taq gene in E.coli, we successfully produced a considerable amount of Taq DNA polymerase with high enzyme activity. Firstly, we extracted plasmids containing the gene encoding Taq polymerase fromE.coli (pTaq1) cells via mini-prep method. Purified plasmids were digested by restriction enzyme EcoRI to produce DNA fragments containing the taq gene, which was then harvested by gel extraction after electrophoresis. Subsequently, taq DNA was ligated to pETBlue-1 vector and transformed into E.coli cells, where the gene was overexpressed. Despite the poor insert gene purity and low transformation efficiency, Taq polymerase was successfully produced in and purified from E.coli, along with high enzyme activity in PCR performance.
Methods
Bacterial cell culture
E.coli (pTaq1) is an Escherichia coli strain that harbours plasmid containing the gene encoding Taq polymerase. E.coli (pTaq1) cells were cultured in L-broth with the antibody carbenicillin at 37°C overnight. Carbenicillin screened out bacteria harbouring the plasmid pTaq1 since this plasmid contains resistance gene against carbenicillin. In our experiment plasmid DNA was extracted from E.coli cultures prepared by the students (culture S) or the demonstrator (culture D). Culture S came from only one single bacterial colony while culture D had a significantly higher cell concentration.
Plasmid DNA extraction
Plasmid DNA was extracted from bacteria cells through Min-prep procedures. Mini-prep isolation protocol is ideal for small plasmid DNA purification and it limits contamination of proteins and genomic DNA [3]. Bacteria cells were harvested from 5 ml of cell suspension by centrifugation. QIA spin prep kits (Qiagen 27104) were used to purified plasmid pTaq1 following the standard procedures. Purified products were separated by electrophoresis in 1% TAE agarose gel containing Sybr Safe dye (InvitrongenTM S33102). The Sybr Safe dye stained the nucleic acids that fluoresced when exposed to UV light. 1kb plus DNA ladder (InvitrongenTM 10787-018) was used to identify the approximate molecular size of DNA. TE buffer and gel loading buffer were made up by students. All gel electrophoresis used the same chemicals in our experiment. Plasmid concentration and purity was determined from A260 and A260/A280 ratio respectively, using a Nanodrop spectrophotometer.
Taq gene purification
To prepare the gene fragment encoding the Taq DNA polymerase, 10μl plasmid DNA was digested by 2μl restriction enzyme EcoRI (New England Biolabs R0101S, 10000units) and incubated at 37°C for 40 minutes. We also included a control reaction in which no EcoRI was added. The pTaq1 plasmid has two EcoRI sites flanking the taq gene and therefore can be digested into two linear DNA fragments. The fragment containing the taq gene has a smaller molecular weight and should migrate faster than the other fragment through agarose gel. After running the EcoRI digest product on an agarose gel we excised the fragment corresponding to the Taq polymerase gene and dissolved the gel slice in threefold volumes of buffer QG in 50°Cwaterbath for 10 minutes. Chemicals and column used to purify taq gene fragment came from the QIAQuick Gel Extraction kit (Qiagen 28704). The concentration of purified DNA was determined by gel electrophoresis as well as Nanodrop spectrophotometer.
Expression and purification of Taq polymerase
For protein expression, taq gene was transformed into E.coli cells using pETBlue-1 plasmid vector. The vector had been previously cleaved with the EcoRI restriction enzyme and treated with alk\(\)\(\)aline phosphatase to prevent re-ligation. The taq DNA fragments with complementary sticky ends [3] were ligated to plasmid vectors by T4 DNA ligase. The ligation reaction was assembled on ice and then incubated at room temperature (22°C) for 20 minutes. After ligation, the recombinant plasmids were transformed into E.coli cells following traditional Cacl2/heat shock transformation procedure. 3μl ligation products, 3μl supercoiled pETBlue-1 plasmid (positive control) and 3μl water (negative control) were added to every 50μl competent E.colicells, incubated on ice for 15 minutes, heat-shocked at 42°C for 90 seconds and then allow to cool on ice for 2 minutes. For each treatment, 200μl bacterial suspension was plated onto an LB agar petri dish containing the carbenicillin, X-GAL and IPTG and cultured overnight. Blue-white screen was used to screen out transformant colonies and identify recombinants. Recombination frequency (RF) and transformation efficiency (TE) were calculated using the following formula:
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\(RF\ \left(\%\right)=\frac{number\ of\ white\ colonies\ \left(cfu\right)}{total\ number\ of\ white\ and\ blue\ colonies\ \left(cfu\right)}100\%\)
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