Patent Application
20200131521
The present invention relates generally to the field of DNA cloning. More specifically, the present invention relates to reagents and DNA cloning method based on homologous recombination for a site directed cloning of a DNA fragment into a vector.
Molecular cloning, or DNA cloning, refers to the process that inserts a DNA fragment into a vector such as plasmid, followed by the transformation into host cells in order to amplify a target DNA fragment or to express a recombinant protein. Since the first success in 1972, DNA cloning has been widely used and rapidly become the basic and critical technique in biology and biotechnology [1]. Due to its importance, a great number of methods has been developed by a multitude of laboratories to improve efficiency and to reduce the cost of cloning.
Restriction enzyme and ligase-dependent method are long considered as a traditional DNA cloning method. This method is involved two steps prior to the transformation process, which includes first using restriction enzymes to digest the DNA fragment of interest and the selected vector; and second, inserting the target DNA fragment into the linearized vector using a DNA ligase [2]. Although this method is simple, inexpensive, and flexible, it has some limitations due to the lack of appropriate restriction-sites, the difference in optimal conditions of restriction enzymes, and the high background of self-ligated vectors. Thus, this method tends to degrade the DNA cloning efficiency.
To improve the efficiency of DNA cloning, several ligase-independent methods and related kits have been developed and currently available on market such as Ligation Independent Cloning (LIC) developed by New England Biolabs (NEB), In-fusion kit developed by Takara, Cold-fusion kit developed by System Biosciences, etc. These methods are based on the presence of homologous sequences in the ends of the target DNA fragment and linearized vector through the activities of specific enzymes.
In the U.S. Pat. No. 7,575,860 entitled, “DNA Joining Method” to David Evans et al. (hereinafter referred to as the '860 Patent), a method for directionally cloning any linear template DNA molecule into any linearized vector is disclosed. In this method, DNA polymerases having exonuclease activity is used to efficiently join one or more linear DNA molecules sharing ends with appropriate complementation [3]. The method of the '860 Patent does not require the ligation step nor the use of carefully controlled conditions as is required in traditional cloning method.
In another U.S. Pat. No. 8,815,600 entitled, “Homologous Recombination Based DNA Cloning Methods and Compositions” to Liu et al. (hereinafter referred to as the '600 Patent′), methods and compositions for cloning a donor DNA molecule into an acceptor vector at a predetermined location are described [4]. The methods of the '600 patents are based on homologous recombination mediated by in vitro enzyme cocktail containing an exonuclease and a single-stranded DNA binding protein.
In general, these methods have advantages over the traditional method such as high cloning efficiency, the digestion of target fragment with restriction enzyme(s), and time-saving. However, because these techniques use purified enzymes, their cloning kits are expensive. Thus, small laboratories hesitate to use them.
In a patent application publication No. 2013/0045508 entitled, “Cell Extract Promoted Cloning” to Eldelmann et al. (herein after referred to as “'508 publication”), a method of assembling a plurality of double-stranded DNA fragments into DNA modules in a single in vitro recombination reaction is disclosed [6]. The chief aims of the method of the '508 publication are to insert a DNA fragment into a vector using cell extracts derived from a RecA-deficient Escherichia coli (hereinafter referred to as “E. coli”) strain and requires homologous sequences in the ends of DNA molecules. There are three different mechanisms of homologous recombination in E. coli. The first is RecA-dependent, the second is RecA-independent, and the third is Red/ET-dependent [7]. RecA-dependent homologous recombination requires long homologous regions (150-300 bp), but it is the main and stronger recombination pathway. RecA-independent recombination pathway does not require long homologous sequence (a minimum of 12 bp) but it occurs at low efficiency in E. coli [8]. Therefore, although this method is easy, efficient, and cost-effective, the use of extract from E. coli cells lacking RecA, an important protein in bacterial repair system, might affect the recombination efficiency and induce mutations during cloning.
Furthermore, it is well known in the art that E. coli does not have tightly regulated DNA-repair mechanism in comparison to eukaryote cells. Therefore, unwanted mutations might occur during combination. On the other hand, in vivo DNA cloning based on yeast homologous recombination has been widely used due to its high efficiency [9]. The required homologous sequence is shorter, approximately 29 base pairs (bp) [10]. Additionally, the yeast recombination has strong mechanism for control of homologous DNA repair, which limits mutations [11]. However, there are no previous work that reportedly used the extracts of Saccharomyces cerevisiae (hereinafter referred to as “S. cerevisiae” or Saccharomyces”) for in vitro DNA cloning. In fact, all intact Saccharomyces used in vivo cloning requires a vector that can replicate independently with yeast chromosome. This requirement limits the types of vectors that can be used.
Therefore, what is needed is a reagent for DNA cloning that is simple and cost-effective so that it can be used by small laboratories.
Furthermore, what is needed is a DNA cloning method that is easy to perform.
Yet, what is needed is a DNA cloning method that is accurate and efficient.
Yet, what is needed is a DNA cloning method that achieves minimal mutations. The present invention provides solutions to the above needs.
Accordingly, an objective of the present invention is to provide novel reagents and a cloning procedure based on homologous recombination for the site-directed cloning of a DNA fragment into a vector at designed site(s). The cloning reagents are made of mixture of extracts from at least two different cell types, preferably a mixture made of extracts from wild-type E. coli and S. Cerevisiae. Due to the activity of the mixture of cell extracts, recombination occurs between the 3′ and 5′-ends of the target DNA and at the appropriate ends of linearized vector, which facilitates in-frame construction of expression vectors.
Another objective of the present invention is to provide an efficient method of using reagents for DNA cloning that is cost-effective, simple to perform, and accurate with minimal mutations, especially for in-frame gene construction.
Yet another objective of the present invention is to provide a reagent in a DNA cloning procedure that will yield efficient and accurate results.
These and other advantages of the present invention will no doubt become obvious to those of ordinary skill in the art after having read the following detailed description of the preferred embodiments, which are illustrated in the various drawing Figures.
The accompanying drawings, which are incorporated in and form a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.
Reference will now be made in detail to the preferred embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention will be described in conjunction with the preferred embodiments, it will be understood that they are not intended to limit the invention to these embodiments. On the contrary, the invention is intended to cover alternatives, modifications and equivalents, which may be included within the spirit and scope of the invention as defined by the appended claims. Furthermore, in the following detailed description of the present invention, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be obvious to one of ordinary skill in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, components, and circuits have not been described in detail so as not to unnecessarily obscure aspects of the present invention.
It will be noted that the cloning efficiency mentioned herein refers to the ratio of colonies bearing the desired DNA fragment, which is inserted into vector in correct position, direction, and without mutation vs. the total number of colonies. The resulting vector from this cloning method might be used directly for expressing the target gene since correct site, proper direction assembly and no mutation are crucial for an efficient gene expression. Although we do not know the exact synergistic mechanism of the cell-extract mixture, it is clear that the yeast extract supported the function of E. coli proteins while controlling DNA repair processes that limit mutations.
As used herein, the term “wild-type E. coli” refers to the E. coli strain isolated from the natural environment and has not been genetically modified.
As used herein, the term yeast refers to the yeast S. cerevisiae. Accordingly, the wild-type yeast is the S. cerevisiae strain isolated from the natural environment and has not been genetically modified, whereas the term “laboratory strains” means the S. cerevisiae strains which has been genetically modified and widely used in laboratories.
As used herein, the term “polymerase chain reaction” (PCR) refers to a technique for rapidly synthesizing a huge amount of copies of a particular DNA fragment based on the activity of a DNA polymerase.
As used herein, the term “cDNA” indicates a DNA synthesized from a single stand mRNA through the action of an enzyme reverse transcriptase.
As used herein, the term “target DNA fragment” or “target fragment” refers to the DNA molecule which is inserted into a vector.
As used herein, the term “recombinant vector” means the vector containing the target DNA fragment.
Many aspects of the present invention are now described with reference to
Method 100 begins at step 101 where DNA fragments are prepared. In one implementation of step 101 of the present invention, the DNA fragments can be produced by amplifying DNA or cDNA using a polymerase chain reaction (PCR) method. Each primer used in the PCR method contains two ends: a 5′ end (or phosphate end) which is homologous to the appropriate end of the vector or another DNA fragment at the assembly site to allow the recombination, and a 3′ end (or hydroxide) which is specific to the target fragment to ensures the successful PCR amplification.
Next at step 102, the plasmid vectors (or recipient cells) are linearized at the selected sites. In one implementation of step 102 of the present invention, the linearized vector can be generated either by digestion with restriction enzymes or by amplification of a circle vector using the PCR method.
At step 103, a reagent including extracts from E. coli strains and yeast strains are prepared. In one implementation of step 103 of the present invention, the E. coli strains are the wild-type E. coli strains. The wide-type strain was isolated from Laboratory of Molecular Biotechnology, VNU-HCM University of Sciences, Viet Nam and has been deposited with the Agricultural Research Service Culture Collection in Peoria, Ill., USA on Jun. 21, 2018 under the Budapest Treaty and was assigned accession number NRRL B-67662. The yeasts can be the wild-type or laboratory S. cerevisiae strains. The S. cerevisiae strains used in the implementation of step 103 are any commercial S. cerevisiae yeasts including, but not limited to, W303, BY4741, BY4742, MT8-1, and YPH250.
Continuing with step 103, in one aspect of the present invention, the E. coli strains having the identifying characteristics of deposit NRRL B-67662 are produced by the following specific steps:
(a) preparing a batch of wild-type E. coli strains overnight at 37° C. in a lubria broth (LB) medium to obtain a E. coli seed culture;
(b) diluting a 1 ml of the E. coli seed culture in a 100 ml fresh medium to obtain a diluted E. coli seed culture;
(c) culturing the diluted E. coli seed culture in step (b) above until an optical density measured at 600 nm wavelength (OD600) value is 1.0 to obtain E. coli cells;
(d) centrifuging the E. coli cells in step (c) above at 5.000 rpm at 4° C. for 10 minutes and then washing them twice with distilled water (dH2O) to collect a pellet of E. coli cells;
(e) suspending the pellet of E. coli cells in step (d) above in a buffer containing 1% SDS, 5 mM of an Ethylenediaminetetraacetic acid (EDTA);
(f) incubating the pellet of E. coli cells in step (e) above at room temperature for 10 minutes to lyse E. coli cells;
(g) removing the E. coli cell debris in step (f) above by a centrifugation at 13.000 rpm and 4° C. for 10 minutes to obtain a supernatant of E. coli cells;
(h) mixing the supernatant of the E. coli cells with an equal volume of 100% glycerol to obtain a mixture of E. coli extract; and
(i) storing the mixture of E. coli extract at −30° C.
Continuing with step 103, any commercially available yeasts such as a W303, a BY4741, a BY4742, a MT8-1, and a YPH250 can be used with the above E. coli to generate the reagent of the present invention. In one exemplary implementation of step 103, a similar protocol with E. coli extract preparation was used to prepare the yeast extract. In brief, the S. cerevisiae W303 strain was grown overnight at 30° C. in a yeast peptone dextrose (YPD) medium to prepare the seed culture. More particularly, the S. cerevisiae W303 strain is prepared by the following process:
(h) growing a S. cerevisiae seed culture overnight at 30° C. in a yeast peptone dextrose (YPD) broth;
diluting a 1 ml of the S. cerevisiae W303 seed culture in a 100 ml of a fresh medium;
(j) culturing the S. cerevisiae seed culture in step (b) until an optical density measured at 60 nm wavelength (OD600) value of 1.0 is achieved;
(k) collecting and washing the S. cerevisiae seed culture twice with distilled water (dH2O) to obtain a pellet of S. cerevisiae cells;
(l) suspending and homogenizing the pellet of S. cerevisiae cells in step (d) in a 50 mM of Tris-HCl pH 7.0 to obtain a pellet of S. cerevisiae cells;
(m) removing debris from pellet of S. cerevisiae cells by a centrifugation process to obtain a supernatant of S. cerevisiae cells;
(n) mixing the supernatant of S. cerevisiae cells in step (f) with an equal volume of 100% glycerol to obtain a mixture of S. cerevisiae extract; and
(o) storing the mixture of the S. cerevisiae extract at −30° C.
It will be appreciated that step 101, step 102, and step 103 are independent. That is, these steps can be performed in any order.
Next, at step 104, the cloning reaction is set up that includes the DNA fragments of step 101, the linearized plasmid vectors of step 102, and the reagent of step 103. In various implementations of step 104, the reaction mixture is allowed to react in vitro for a desired time at a desired temperature: the desired time ranges from 5 min to 2 days and the desired temperature ranges from room temperature to around 50° C. In a preferred implementation of step 104, the reaction conditions are 30 to 40° C. for 15-30 minutes when the length of each of the inserted DNA fragments is less than or equals to (≤) 1,000 base pairs (bp) and that of each of the DNA plasmid vectors is less than or equals to (≤) 7,500 bp. On the other hand, the reaction temperature is set at 37 to 45° C. for 30-60 minutes if the length of each of the DNA fragments is at least 1,000 bp and that of each of the DNA plasmid vectors is at least 7,500 bp.
Next at step 105, the recombinant DNAs obtained from step 104 are transformed into host cells. In various implementations of step 105, the recombinant DNA from the cloning reaction can be transformed into E. coli competent host cells using standard transformation methods in order to select and amplify recombinant vectors.
In one exemplary implementation, the recombinant DNA is introduced into the E. coli host cells if the lengths of the inserted DNA fragments and plasmid vectors are less than 2,000 base pairs (bp) and 10,000 bp respectively. As such, the E. coli host cells are cultured in an appropriate medium that can enable a quick screening process based on the resistance of antibiotics genes or α-complementation of the β-galactosidae genes following by the colony from PCR with specific primers. Finally at step 106, the result recombinant DNAs achieved from step 105 are screened to select the desired DNA cloning colony and store them at −30° C.
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In the present invention, the insertion one or several DNA molecule(s) into a vector at designed site(s) relies on enzymes provided by a mixture of cell extracts from selected S. cerevisiae and E. coli strains. The cell extract mixtures allow in vitro cloning with short homologous sequence length (˜15 bp). Advantageously, the E. coli strains used in this invention is preferentially of wild type, contrarily to previously patented work that were limited to specialized RecA-deficiency bacteria strains. Prior to this invention, no one used RecA-dependent or wild type E. coli (which has RecA) cells for either in vivo or in vitro cloning methods.
This invention will be better understood from the Experimental Details and Results. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.
Materials and Methods
Strains and Medium
The E. coli strain used for extract preparation was isolated by Lab. Molecular Biotechnology, University of Science—Ho Chi Minh city. The S. cerevsisiae W303 strain was used to prepare the yeast extract. E. coli and S. cerevisiae cells were cultured in LB (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) and YPD (10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose) media, respectively.
Plasmid, Insert fragments and Primers
The backbone plasmid pET-28a(+) (Novagen) was used in all cloning experiments. For cloning one DNA fragment, the DNA fragments with different sizes were synthesized and named as ES1 (300 bp), ES2 (1000 bp), ES3 (1500 bp). Primers for DNA amplification were designed as following: the 5′-end containing 10, 15, or 20 nucleotides homologous to the appropriate end of the vector/DNA fragments it will join; and the 3′-end containing 20 nucleotides specific to the target DNA fragment. Please refer to
E. coli Extract Preparation
The E. coli strain was grown overnight at 37° C. in LB medium to prepare the seed culture. Cells from 1 ml seed culture were diluted in 100 ml fresh medium and cultured further until they reached OD600=1.0. Cells were collected by centrifugation at 5.000 rpm, 4° C. for 10 minutes and washed twice with dH2O. The pellet was suspended in buffer containing 1% SDS, 5 mM EDTA (1 g cells in 10 ml buffer) and then incubated at room temperature for 10 minutes to lyse cells. Cell debris was removed by centrifugation at 13.000 rpm, 4° C. for 10 minutes. The supernatant was mixed with an equal volume of 100% glycerol and stored at −30° C.
Yeast Extract Preparation
A similar protocol with E. coli extract preparation was used to prepare the yeast extract. In brief, the S. cerevisiae W303 strain was grown overnight at 30° C. in YPD medium to prepare the seed culture. Cells from 1 ml seed culture were diluted in 100 ml fresh medium and cultured further until they reached OD600=2.0. Cells were collected and washed twice with dH2O. The pellet was suspended and homogenized in 50 mM Tris-HCl pH 7.0 (1 g cells in 10 ml buffer). Cell debris was removed by centrifugation and the supernatant was mixed with an equal volume of 100% glycerol and stored at −30° C.
Cloning Protocol
The vector was linearized using the restriction ezyme BamHI. DNA fragments were amplified using specific primers containing different length of homologous sequences at their 5′ ends. A 15 μl reaction of 100 ng linearized plasmid, 30 ng DNA fragment, 1.5 μl buffer (50 mM Tris-HCl, 20 mM MgCl2, 2 mM ATP, 20 mM DTT, pH 7.0 at 25° C.)_and 1 μl extract mixture was prepared and incubated at 37° C. for 15 minutes to allow the homologous recombination. After that, 10 μl reaction mixture was transformed into 100 μl E. coli DH5α competent cells using classical calcium chloride (CaCl2) method [12]. The transformed cells were plated on LB agar plates containing with 30 μg/L Kanamycin. Positive recombinant clones were selected by PCR with specific primers. The sequence accuracy of inserts was verified by DNA sequencing. All experiments were repeated at least 3 times.
Expression Verification
Several recombinant clones were randomly chosen and grown in 5 ml LB medium at 37° C. until they reached OD600=0.5. After that, 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added and cells were further cultured for 2 hours. Cells from 1 ml of each culture were collected by centrifuge, then suspended in 200 μl SDS-PAGE loading buffer (50 mM Tris-HCl, 2% SDS, 0.1% bromophenol blue, 10% glycerol, and 100 mM dithiothreitol) and incubated at 100° C. for 20 minutes. Ten microliter (μl) sample was applied to each lane of a 12.5% polyacrylamide gel for SDS-PAGE analysis. The 6×His-tagged proteins were detected by western blotting using a monoclonal anti-Histidine antibody (sc-8036, Santa Cruz biotechnology).
Combination of E. coli Extract and Yeast Extract Exerts a Synergic Effect on DNA Cloning
It has been previously reported that E. coli extract has ability to assemble a linearized vector and a DNA fragment based on the presence of homologous sequences on them. In addition, an in vivo cloning method based on homologous recombination of intact yeast cells has been widely used due to its high efficiency and short homology requirement. These backgrounds prompted us to investigate whether the combination of E. coli extract and yeast extract shows a better effect on DNA cloning. Therefore, we performed cloning experiments using different combination ratios of these extracts to insert the ES2 fragment into the vector pET-28a(+). The homologous sequences containing 15 nucleotides was used in this experiment.
The E. coli extract but not the S. cerevisiae extract could work solely to combine the ES2 fragment and linearized pET-28a(+) (Table 2). Importantly, the mixture of 80% E. coli extract and 20% yeast extract (v/v) yielded the better efficiency, in which the number of transformed colonies and positive ratio were 38.0±6.2 and 96.7±5.8%, respectively. These data suggest that the combination of yeast extract and E. coli extract exerts the synergic effect on DNA cloning. However, the increase in yeast extract concentration in mixture gradually decreased the DNA cloning efficiency. This result might be explained by the dilution of E. coli extract components that negatively affects DNA cloning.
Due to the high efficiency, the combination 80% E. coli extract and 20% yeast extract was used in further experiments. See Table 1 below.
E. coli
Effect of Homology Length on Cloning Efficiency
Since the frequency of homologous recombination is dependent on the homology length and insert size [13, 14], we next investigated the effect of these factors on the extract mixture-mediated cloning. Three genes with different sizes ES1, ES2, and ES3 were used in these experiments. Each gene was amplified by PCR using 3 primers pairs distinguished by the homology length at their 5′-ends. The results showed that the increase in insert sizes obstructed the DNA cloning whereas the increase in homology lengths improved cloning efficiency. Accordingly, the homologous sequence containing 10 nucleotides was sufficient for cloning of ES1 fragment whereas the longer homologous sequences, at least 15 nucleotides and 20 nucleotides, were required for efficient cloning of ES2 and ES3 fragments, respectively. See Table 2.
DNA Sequencing Results Showed High Sequence Accuracy
To examine whether this cloning method induces mutations, 10 clones of each experiment were randomly chosen for DNA sequencing. The results showed that 100% clones contained correct insert sequences. See Table 3. This demonstrated that the extract mixture-mediated cloning is a highly accurate method.
The Extract Mixture-Mediated Cloning is a Site-Directed Method
In the next step, whether the target DNA can be inserted into the correct site in plasmid based on DNA sequencing data is determined. The results show that 100% recombinant plasmid bearing target DNA fragments at the proper positions and correct direction. See Table 4 below.
The Extract Mixture-Mediated Cloning Method is Suitable to Construct Expression Plasmids
All examined DNA fragments in this invention were designed and inserted into plasmid pET-28a(+) at EcoRV site and in-frame with 6×His-tag sequence so that they can be expressed under the control of T7 promoter. Therefore, in order to verify gene expression, several recombinant plasmids were randomly chosen and transformed into E. coli BL21(DE3). The presence of target proteins was detected by SDS-PAGE and Western Blot using anti-Histidine antibody since the proteins were fused with 6×His-tag. The representative SDS-PAGE and Western Blot results were shown in
Examining the Stability of Extract Mixture Under Several Storage Conditions
For the purpose of product commercialization, the stability of extract mixture during storage at 25° C. (room temperature), 10° C. (refrigerator temperature), −30° C. (freeze temperature), and −80° C. (deep freeze temperature) was examined.
At 25° C., the activity of extract mixture rapidly decreased after the first day of storage is shown in graph 500A of
In the present invention, the extract from wild-type E. coli cells could allow the in vitro DNA assembly. More importantly, the combination of 80% wild-type E. coli extract and 20% yeast extract demonstrated a synergic effect on DNA cloning. Using the extract mixture, the cloning one or multiple DNA fragments up to 1500 bp long containing 10-20 bp homologous sequence into vector at proper sites and correct direction was successful. Since this cloning method is not depended on DNA ligase activity, the ratios of positive clones to all obtained clones are relatively high, that can speed up the subsequent screening and verification steps. In addition, as discussed above, unlike the previous related inventions that did not mention gene expression, the present invention showed that all tested recombinant clones could express target proteins, suggesting the improvement of the present method in constructing expression vectors. Therefore, the cell extract mixture of the present invention, which can be maintain its DNA assembly activity for at least one year at below −30° C., has a great potential for the development of an easy, cost-effective, accurate and site-directed DNA cloning kit that the market long awaits for but could not achieve.
The foregoing description details certain embodiments of the invention. It will be appreciated, however, that no matter how detailed the foregoing appears in text, the invention can be practiced in many ways. As is also stated above, it should be noted that the use of particular terminology when describing certain features or aspects of the invention should not be taken to imply that the terminology is being re-defined herein to be restricted to including any specific characteristics of the features or aspects of the invention with which that terminology is associated. The scope of the invention should therefore be construed in accordance with the appended claims and any equivalents thereof.
This application is based upon and claims priority to U.S. provisional patent application 62/707,368 entitled “Mixture of Cell Extract for Site-Directed Cloning” filed Oct. 31, 2017, the entire content of which is incorporated herein by reference.
