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After transformation of fungi in the 'Gene Knock-outs inÂ Trichoderma reesei' experiment, analysis of the relevant transformants by different techniques is necessary to assess if the desired trait was integrated in the transformant. This can be done by performing tests for enzyme activity or by polymerase chain reaction (PCR) based techniques.
PCR is also frequently used for microbial identification and diagnosis. For filamentous fungi like T. reesei, the DNA is often isolated before PCR because the process of DNA extraction reduces a lot of unknown substances present in the biological material that interfere with the PCR process. In this exercise, a procedure for rapid mini preparation of fungal DNA for PCR is introduced. Such DNA extraction procedure is simple, that it does not rely on the use of a grinder with liquid nitrogen for initial breaking up of the mycelia. The presence of mycelia is a significant interference when dealing with a large number of samples.
The goal of this exercise is to determine the success of transformation of T. Reesei KU70 strain using PCR.
Figure 1.1 shows the potentialÂ T. reeseiÂ KU70 transformants transferred to PDA media plates in the previous experiment (Gene Knock-outs inÂ Trichoderma reesei). DNA isolation was done using the rapid mini preparation of fungal DNA procedure. Purified DNAs were loaded on agarose gel to confirm the presence and size of obtained DNAs.
Figure 1.1.Â Potential T. reesei KU70 transformants on PDA plates.Â
PCR assay of purified DNAs was done in parallel with the agarose gel electrophoresis. Deviations from the protocol were done by adding 2 ÂµL of DNA (instead of 1 ÂµL) and adding 5 ÂµL MgCl2 for the master mix. PCR products were loaded on agarose gel to confirm the presence and size of DNAs to consequently evaluate results of transformation.
From the 'Gene Knock-outs inÂ Trichoderma reesei' experiment, gene pyr4 was integrated to T. reesei KU70 strain. The obtained transformants were analyzed by PCR in this exercise to see if the construct was integrated by homologous recombination. Â
Figure 1.2 shows the result of agarose gel electrophoresis of T. reesei KU70 transformants. The success of transformation is indicated by high DNA size. Wild type DNA is assumed at ~3000 bp, while transformants are expected to have ~5000 bp. Strains 1, 9, 12, 13, 14, and 17 show bands at ~5000 bp, indicating the presence of transformants. Very strong bands are present in lower size level, which are presumed to be degraded DNA and/or by-products. Strains 1 and 9Â show the presence of both bands (wild type and transformant). This indicates that not only the transformant is present, but also a background (i.e. protoplast from the agar might have been taken as well). To avoid the presence of this background, more appropriate primers can be chosen or isolation of single spores can be done by streaking spores from PDA plates to another plate.
Figure 1.2. Agarose gel electrophoresis of T. reesei KU70 transformants.
After transformation, selection, and purification, transformants can be analyzed for stable integration of the transgene by PCR-based methods. Normally, a first PCR screen of the transformants is based on the detection of stable integration of the marker gene. For verification of integration of the construct, primers are chosen based from sequences of the marker cassette and from the gene of interest. Upon using appropriate primer combinations, PCR products are obtained when the construct is integrated at the genomic locus of interest by means of homologous recombination. With the use of such primers, the size of the PCR product will change according to the size of the inserted selection marker cassette indicating the occurrence of homologous recombination.Â
Homologous recombination involves the breaking and re-merging of two double strands of DNA with closely-related sequences. This process allows the integration of genes into particular genomic loci. In transformations using circular plasmids, the entire plasmid is incorporated into a chromosome through a single cross-over. On the other hand, a double cross-over involves a linear DNA molecule in the replacement of a chromosomal gene by a gene with similar sequences to the chromosomal gene at the cross-over regions.
Figure 1.3 shows the specific gene deletion in the T. reesei KU70 strain. Upstream (5') and downstream (3') regions that are immediately next to the chromosomal gene to be deleted (Gene) are integrated into a vector that consists of a suitable selection marker (pyr4). The circular DNA or plasmid is converted to a linear DNA with the aid of a restriction enzyme that cuts the plasmid once. This linearized vector then undergoes transformation into a pyr4 mutant strain. A double cross-over or double homologous recombination occurs in the 5' and 3' regions, replacing "Gene" in the chromosome with the "pyr4" selection marker.
Figure 1.3. Specific gene deletion in T. reesei KU70.
In conclusion, the result of this exercise confirmed the transformation in T. reesei KU70 strain by using a PCR-based method.
Strain Improvement by Recombinant DNA Technology
Most organisms produce enzymes economically and are usually in small amounts. To increase yields of these practically important enzymes, an easy method to improve a certain strain can be employed through the technique of recombinant DNA technology.Â
Plant cell walls are mainly composed of cellulose, hemicelluloses and pectins. Pectin is a structural complex heteropolysaccharide present in the primary cell walls and middle lamella of plants. Pectins have Dâ€galacturonic acid (Dâ€GalA) in their backbone and the linear pectin polymer homogalacturonan (HG) consists of Î±â€1,4â€linked Dâ€GalA backbone with different small side groups. With more complicated side chains are other pectic structures such as rhamnogalacturonan I and II (RGâ€I, â€II), xylogalacturonan (XGA), and apiogalacturonan (AP). The fungi Trichoderma reesei is known as an effective producer of numerous plant cell wall degrading enzymes such as cellulases and xylanases for numerous biotechnological applications. Among these enzymes, only one pectinase (endopolygalacturonase) is found to damage the backbone of homogalacturonan, but this enzyme is weakly produced.
This experiment aims to overexpress the endogenous endopolygalacturonase (endo-PG) pec1 in Trichoderma reesei TU-6 strain to improve the pectin degrading ability of the fungus. The pectinase structural gene will be cloned under a strong constitutive promoter (pyruvate kinase promoter) and will be transformed into T. reesei. The overexpression of the endopolygalacturonase gene in the transformants will then be tested by a plate assay which measures the enhanced pectin degradation.Â
This experiment was done in parallel with the 'Gene Knock-outs in T. reesei' experiment. Spore solutions of the T. reesei TU-6 strain was prepared. A solution of NaCl/Tween 80 was used to harvest the spores, where the Tween helps to better solubilize the hydrophobic fungal spores in the solution. Spore solutions were then inoculated on several MEX-U plates with cellophane discs. The plates were then incubatedÂ at 28Â°C for ~20 hours.
After incubation, freshly grown mycelium on cellophane discs were used for protoplasting. Producing protoplasts involves the removal of fungal cell wall with lytic enzymes. Protoplasts are commonly used for DNA mediated transformation because the presence of cell wall would block the passage of DNA into the cell. The quality of the protoplast suspension was inspected under the microscope, but counting of the protoplasts in a Thoma counting chamber was not done.
For the transformation, the protoplast suspension was mixed with the purified DNA fragment (pec1) and polyethylene glycol (PEG). In this procedure, the protoplast is able to take up DNA in the presence of CaCl2 and PEG. The resulting solution was distributed to several plates of minimal medum with sorbitol, also using overlay medium. The plates were then incubated for 5 days at 28Â°C.
After incubation, potential transformants were transferred to selection plates. After 6 days, spores were transferred to minimal medium plates containing polygalacturonic acid.
Potential transformants were inoculated on test plates containing polygalacturonic acid. After 3 days, the plates were treated with an aqueous solution of ruthenium red and thereafter rinsed with distilled water. Areas of polygalacturonic acid degradation were identified by evaluation of intense purple-red halo around fungal colonies.Â Â
Potential transformants of the theÂ T. reeseiÂ TU-6Â strain were tested by a plate assay which measures the enhanced pectin degradation or the overexpression of endo-PG on the strain. Figure 2.1 shows the result of this assay. On plate A is colony 1, the control, which possesses an overexpression of endo-PG. Comparing the grown colonies to colony 1, colonies 2 (plate B) and 3 (plate D) show large radius of intense purple-red halos. This signifies the two colonies as potential transformants with enhanced endo-PG activities.Â
Figure 2.1.Â Plate assay of enhanced endopolygalacturonaseÂ
activity ofÂ T. reesei TU-6 strain.
Ruthenium red used in this assay penetrates beneath the surface layers of the agar, in the regions around a fungal colony where degradation of polygalacturonic acid occurred. When there is no degradation of polygalacturonic acid, ruthenium red does not penetrate the medium. If this is the case, it is restricted to binding to the surface layers and is easily washed off. Parts of the medium with undegraded polygalacturonate show a colourless clear background and areas with polygalacturonic acid degradation show an intense purple-red halo. The method is commonly used to screen yeasts and filamentous fungi for secretion of polygalacturonase.
In enzyme production, it is essential to produce yields that are economically viable and highly pure. In some instances, yields that are 1000 times higher than those produced by the original source are necessitated. Â In this case, gene technology plays an important role.Â For an optimal production strain, a most suitable vector is required. The majority of expression vectors are plasmids or DNA fragments from plasmids, which are incorporated into the genome of host organisms. Shown in Figure 2.2 is the expression vector used in this exercise, where it consists of a selection marker (pyr4) and an expression cassette that encodes the enzyme gene of interest. The expression cassette contains the enzyme gene of interest (pec1), a promoter (Ppki1) to direct transcription of the gene that encodes the enzyme of interest, and a terminator of pec1 (Tpec1). The efficiency of the expression vector is mainly influenced by the amount of functional messenger RNA produced from the complex relationship between the promoter, the structural gene, and the host strain.
Figure 2.2. Expression vector for endo-PGÂ pec1 gene.
After assembly of the expression vector containing the enzyme gene, it is then transferred into the host strain by transformation, where the host cells are treated by chemicals or enzymes to make the cell walls accessible to the expression vector DNA. After incubation of cells in the presence of the expression vector, the cells regenerate cell walls and placed on a selective growth medium that permits the growth of only those cells that have integrated the expression vector with its selection marker and make way for its stable replication. These are now the resulting transformants, which are then tested for the enhanced ability to produce the recombinant enzyme. Â
Microbial pectinases, the endo-type in particular, play a vital role in the food industry due to the fact that they favor the extraction, clarification and reduction in viscosity of fruit juices. Production of these enzymes in most microorganisms is, however, limited by mechanisms which regulate their synthesis. Â The majority of pectinases are induced by pectin and are prone to repression because of the presence of repressor substances or of products related to the degradation of pectin as reported for some polygalacturonases in other filamentous fungi. Currently, the microbial synthesis of enzymes at industrial level needs highly productive strains to reduce production costs. Thus, the use of microorganisms resistant to catabolic repression could diminish enzyme repression and therefore increase production yields in microbial fermentation. Fungi are preferred in the production of industrial pectinases, since these may be excreted into the culture medium and ease of isolation is possible.
To conclude, the strain improvement of T. reesei TU-6 was achieved as proved by the assay, showing qualities of overexpressed endo-PG.
Fusion/Overlap Extension PCR
In constructing gene expression or gene deletion cassettes, a number of time-consuming cloning steps are usually performed. The typical method depends on the number of subcloning and ligation steps, the availability of appropriate restriction sites, and on the successful intermediate subcloning into a suitable plasmid. This method is done in a stepwise procedure that involves PCR amplification, restriction enzyme cleavage of the gene fragments, and ligation of the different fragments in an appropriate restricted cloning vector. Ligation assay is then transformed into Escherichia coli for amplification and verification of the plasmid construct.
Due to the tediousness of the conventional method, a more effective and practical characterization of genes is preferred using an easy and fast procedure for building constructs. For this purpose, overlap extension polymerase chain reaction (PCR) as a tool for vector construction is often being used. This PCR technique functions in replacing genes, tagging genes with fluorescent markers or epitope tags, or replacing endogenous promoters with inducible ones and introduce a suitable selective cassette.
The objective of this exercise is to use the technique of fusion PCR to successfully fuse two gene fragments into a single molecule.
A first PCR was performed to amplify the 4 gene fragments (gene A1, gene B, gene A2, gene C) to be used in making 2 constructs.
Â Â Â Fusion construct 1:
Â Â Â Â Â Â Gene A1 fragment (520 bp)Â
Â Â Â Â Â Â primer 1: 5' CAGCTCACGTAAAGCGTCATGTTGTCCAATCCTCTCCGTCG 3'
Â Â Â Â Â Â primer 2: 5' CACGACTGCGGATAGGTCTCTTGCGCGAAGCTTTGCC 3'
Â Â Â Â Â Â Gene B fragment (2575 bp)
Â Â Â Â Â Â primer 3: 5' GGCAAAGCTTCGCGCAAGAGACCTATCCGCAGTCGTG 3'
Â Â Â Â Â Â primer 4: 5' CGCGGATCCGCGACCCAGAGCTGAGGAAGAG 3'Â BamHI
Â Â Â Fusion construct 2:
Â Â Â Â Â Â Gene A2 fragment (559 bp)
Â Â Â Â Â Â primer 1: 5' CAGCTCACGTAAAGCGTCATGTTGTCCAATCCTCTCCGTCG 3'
Â Â Â Â Â Â primer 2: 5' CACGACTGCGGATAGGTCTTTGTGCTGCAGCCGCC 3'
Â Â Â Â Â Â Gene C fragment (2573 bp)
Â Â Â Â Â Â primer 1: 5' GGCGGCTGCAGCACAAAGACCTATCCGCAGTCGTG 3'
Â Â Â Â Â Â primer 2: 5' CGCGGATCCGCGACCCAGAGCTGAGGAAGAG 3' BamHI
Â Â Â Â
The grey-highlighted parts of the oligonucleotides correspond to gene A, the underlined parts are the restriction sites, and non-marked parts are gene B or C specific.
The first PCR products were loaded on agarose gel to confirm the presence and size of DNA fragments. The fragments were then cut out of the gel and purified using gel extraction kit. Purified DNAs were again laoded on agarose gel to confirm the presence and size of DNA fragments. Using NanoDrop, the concentrations of purified DNA were determined. These concentrations were used in calculating the ratio of DNA fragments to be added for the second PCR.
The second PCR was performed to fuse the different fragments (construct 1: gene A1 + B, construct 2: gene A2 + C) by hybridization of their overlapping ends. This was done in a two-step procedure where the DNA fragments were added for PCR assay, but first without primers, just to fuse the fragments into a single moleceule.Â After this run, outer primers 1 and 4 were then added for amplification of fused fragments. Finally, PCR products after the second PCR was loaded on agarose gel to evaluate the success of fusion PCR of 2 constructs by confirming the expected sizes.
All PCR assays were done using Phusion DNA polymerase.Â Phusion DNA polymerase is a proofreading enzyme with enhanced DNA-binding activityÂ and processivity and has the lowest error rates of any polymerases available.
Figure 3.1 shows the result of agarose gel electrophoresis after the first PCR, where each gene fragment (gene A1, C, A2, and B) was amplified. This result confirmed the sizes of the fragments as well. The strongest bands show the fragments of interest. The other light bands are unwanted DNA fragments. Thus, a subsequent DNA purification was performed.
Figure 3.1. Agarose gel electrophoresisÂ
Â Â Â Â of first PCR products.
After DNA purification, the products were again run on an agarose gel as shown in Figure 3.2. It can be noticed that the unwanted DNAs were somehow eliminated, except for a light band/smear on genes C and B.
Figure 3.2. Agarose gel electrophoresisÂ
Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â of purified DNA products from first PCR.
The fusion constructs were formed such that gene A1 and B were fused to form fusion construct 1, and gene A2 and C were fused to form fusion construct 2. Theoretically, the fusion constructs should produce fragments with 3095 bp (fusion construct 1) and 3132 bp (fusion construct 2). Figure 3.3 verifies this, showing bands of the fusion constructs at approximately 3000 bp.Â
Figure 3.3. Agarose gel electrophoresisÂ
Â Â Â Â Â of fused PCR productsÂ
Â Â after second PCR.
The development of recombinant DNA technology has been in parallel with the construction of Escherichia coli plasmid vectors with DNA sequences that are required for a range of applications in molecular biology. These sequences consist of drug-resistance genes, E. coli replication origins, promoters, terminators, expression markers, and multicloning sites.Â Restriction digestion of DNA and successive ligation to desired sites within vectors is a conventional recombinant DNA method. Â On the other hand, fusion of DNA fragments can be done by overlap extension PCR. Overlap extension PCR is being used for recombinant DNA constructions, for site-directed mutagenesis, and for cloning of fused segments. The process of PCR-mediated DNA fusion is defined as the association of two DNA fragments with homologous ends during denaturation-reannealing, followed by extension by polymerase reaction. The mechanism of this method as performed in this exercise is shown in Figure 3.4. In the first PCR step (PCR 1), the 2 DNA fragments to be fused (i.e. gene A1 and B) are amplified using appropriate primers (primers 2 and 3), whose 5' ends partially match a part of the adjacent fragments in the final vector construct. In the second PCR step (PCR 2), the fragments from PCR 1 are finally fused by hybridization of the overlapping parts of the different fragments and the strands are filled in by the polymerase to form the final fusion construct. The same scheme is followed for producing the fusion of gene A2 and C.
Figure 3.4.Â Schematic diagram of Fusion PCR.
In theory, overlap extension PCR is a straightforward procedure but it has not been frequently used when compared with recombinant plasmid construction. Reasons for this are the technical requirements for the adjustment of PCR conditions, the cleanliness of the initial DNA template, and the accurate design of primers. The high purity of the initial DNA template and the accurate design of primers have been presumed to be crucial for successful overlap extension. Nevertheless, the construction of recombinant DNA constructs by PCR overlap extension would be a very functional tool for genome-wide analyses using large numbers of DNA fragments. To more easily apply overlap extension PCR to the generation of conventional DNA vectors, intense annealing of overlapping sequences of target fragments should occur under simple PCR conditions.
Developments in PCR overlap extension have occurred in recent years, such as long multiple fusion, allowingÂ the fusion of multiple fragments rather than only two fragments.Â This new method provides molecular biologists much wider opportunities in the design of recombinant DNA. With the existing methodologies, some of the things that would be impossible includeÂ the generation of somatic cell knockouts for a wide range ofÂ human genes, generation of multiple custom-made viral genomes, and a number of transgene experiments. This is mainly due to limitations that result from the lack ofÂ suitable restriction sites at junctions or the multiple occurrences of otherwise usefulÂ restriction sites in the case ofÂ long DNA constructs, which cause them to be useless. For these reasons, researchers are usually forced to assemble constructsÂ that include unwanted DNA sequences using current cloning methods. Applying methylation, partial restrictionÂ digestion, recombinase, adapters and linkers can minimize some of these problems, making a taskÂ more complicated and time-consuming. These complications are overcome by the method of long multiple fusion. This method is expected to significantly make other complex genetic engineering projects possible.
In this exercise, it can be concluded that two rounds of PCR (fragment amplification and fusion) can produce recombinant DNA products by fusion PCR of two DNA fragments. This method is an easier method than the conventional restriction and ligation. Overlap sequences are potentially important to contribute an extensive application in the design and production of DNA constructs for different molecular and genetic analyses.