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Trichoderma reesei (anaphorm of pantropical ascomycete Hypocrea jecorina) is the filamentous fungus that is considered as one of the most important (industrial-wise) organisms used in biotechnology for the production of cellulases and xynalase. Some strains (mainly the'industrial workhorses') can reach very high protein production levels, even 100 g/l 1. The strains of T. reesei that are nowadays used on the industrial scale were originally obtained during the Second World War on the Salomon Islands from just one isolate 2,3.
The size of T. reesei genome is 34Mb and it only contains seven chromosomes 4
T. reesei is generally considered as non- pathogenic for people 5,6 therefore cellulases produced by strains of Trichoderma are regarded as safe for use by man.
The incredible capacity and effectiveness of this fungus to secrete such large amounts of enzymes (cellulases and hemicellulases) surprisingly does not find reflection in T. reesei genome. In fact, Trichoderma among all other fungi was discovered to have the smallest number of genes for enzymes taking part in hydrolisation of plant cell walls polysaccharides (pectinases, cellulases, hemicellulases) 4.
In our experiment we used the Trichoderma reesei mutant strain Rut-C30. This hypercellulolytic strain was first isolated by D. Eveleigh and B. Montenecourt 7,8 and since then has become one of the most widely used strains among those employed for laboratory enzyme (cellulase) production 9,10. The Rut-C30 needs only one carbon source to grow and it is able to produce cellulases as well as xylanases. This particular strain was found to have a truncation in the cre1 (carbon catabolite repressor) that encodes CRE1, which in turn makes it carbon catabolite depressed 11. The second known genetic difference in the Rut-C30 strain compared to other strains is the mutation in the gene encoding β-glucosidase II 12. The expression of cellulase in this particular strain was showed to be repressed by glucose in a lower degree as compared to other analysed strains 13.
The aim of our experiment is a genetic transformation of intact conidia of Trichoderma reesei mutant strain Rut-C30, with the aid of Biolistic Gene Gun (PDS-100/He system) with a single barrel with the pHEN54RQDsRed plasmid.
The above mentioned plasmid is equipped with a gene that encodes DsRed1- E5 protein (the Fluorescent Timer), which is a mutant of DsRed1 protein. The wild type of DsRed originates from Discosoma sp.14 The variant of DsRed protein that was chosen for our experiment has increased fluorescence intensity thanks to two amino acid substitutions. It also changes its colour as it gets older 15. When the protein is synthesised it emits green light, but as it gets older, its fluorophore is subjected to some modifications that in result change its fluorescence wavelengths to longer ones. Thus, the protein is bright red when it becomes fully mature.15
In our plasmid, the DsRed1-E5 gene is expressed under the cbh1(cellobiohydrolase I) promoter whereas the selection marker for transformations-the bacterial gene hph (that confers resistance to hygromycin antibiotic because it encodes a hygromycin B phosphotransferase) is expressed under the Trichoderma phosphokinase promoter.
Materials and Methods
Microbial strains and plasmid.
Trichoderma reesei strain Rut-C30 was used in the experiment as a material for the genetic transformation. The DNA plasmid that was used for the transformation was pHEN54RQDsRed containing the gene encoding DsRed1-E5 protein. The gene was amplified from the pTimer plasmid (Bd Biosciences Clontech, USA). The selection marker for fungal transformations was the hph bacterial gene that confers resistance to hygromycin B antibiotic.
Preparation for bombardment.
The conidia were first harvested from a plate in 5ml of 0.9% NaCl, 0.01% Tween 80
Solution. The suspension was then filtered through glass wool and the conidia were counted with the aid of haemocytometer. At this point, a control plate was prepared by spreading 20 ïl of the prepared suspension onto PDA + Hygromycin B.
Next, one portion of conidia (200ïl) was plated in the centre of a plate containing PDA and was then left to dry on the laminar flow.
Precipitation of DNA onto tungsten particles
Around 40 mg of M10 tungsten particles with a mean diameter of 0.6 ïm were washed 3 times in an Eppendorf tube with 1ml of absolute EtOH. Then the particles were rinsed out using distilled water- the procedure was carried out twice. After rinsing, the particles were resuspended in water (1ml) and split into samples each containing 50 ïl of tungsten. Then, 2 ïg of pHEN54RQDsRed DNA were added to the tube with tungsten particles and vortexed for 1minute. Vortexing was continued while 50 ïl of CaCl2 (2.5M) was added followed by 25 ïl of fresh Spermidine (0.1M) to help with precipitation. The solution was left on ice for approximately 10 minutes and after that it was centrifuged for 10 seconds. The supernatant was discarded whereas the pellet was resuspended in 250 ïl of absolute EtOH and centrifuged again. Finally, after removing the supernatant after the second spin, the particles were thoroughly resuspended in 50 ïl of absolute EtOH and left on ice.
The DNA-coated tungsten particles (10ïl) were loaded onto a macrocarrier disc that was previously treated with ethanol for sterilisation, and left to dry. The launch assembly was performed according to the manufacturer's instructions for this particular system. The plate with conidia was located in the chamber at a target distance. The high purity helium pressure was 650 psi and the vacuum was set at 28-29'' Hg. The plate with no DNA served as a negative control for this experiment.
The plate was then incubated o/n at room temperature and layered with 10ml of PDA that contained hygromycin B (3.8 ïl).
Confirmation and counting of transformants
The transformant colonies were counted and transformation efficiency was calculated.
For a second round of selection, a few colonies from PDA+ hygromycin B plate were picked and replated onto a fresh PDA plate containing hygromycin (60U/ml).
Also, a fresh plate was prepared for DNA isolation where colonies from the PDA+ hygromycin B plate were restreaked onto a PDA dish covered with cellophane.
Isolation of DNA
The colonies that grew on the plates after the second selection were scraped off the plates and placed in a mortar and grinded to a fine powder with the aid of liquid nitrogen. Next, the lysis buffer solution (containing 50mM Tris-HCl, 50mM EDTA, 3% SDS and 1% 2-mercaptoethanol) in the amount of 750 ïl was added to the mycelia and incubated at 65oC for one hour. Then a two phase separation was performed by first adding 700 ïl of chloroform: phenol (1:1) , centrifuging at 13,000 x g (15minutes) and removing the aqueous phase to a fresh tube. The second step included the addition of 700 ïl of SEVAG (chloroform: isoamyl alcohol; 24:1) to the previously separated aqueous phase and microcentrifugation for 5 minutes (13,000 x g). Both steps were performed in a fume hood. The aqueous phase was then again removed to a new tube where 20 ïl of sodium acetate were added (3M) followed by 1ml of isopropanol. The solution was microcentrifuged briefly and the pellet was drained. Then , the DNA pellet was washed twice using 500 ïl of 70% ethanol and left till dry at 50oC. After that, the pellet was resuspended in 150 ïl of TE and prepared for the quality check.
The quality check
The concentration and purity of DNA (1:100 dilution of DNA) in the sample was established using a spectrophotometer with readings at O.D 260/280 and O.D 260/230. The quality of the chromosomal DNA was determined by agarose gel electrophoresis with GelRed staining.
After the agarose gel was prepared, 7 ïl of chromosomal DNA were mixed with 6 x sample loading buffer (0.75 ïl) and it was then loaded onto the gel.
The chromosomal DNA from the non-transformant strain was also loaded and served as a control.
PCR reaction and visualisation of the product
The master mix for the reaction was prepared beforehand and contained:
34.75 ïl of water
5 ïl of 10 x Taq Buffer
1 ïl of 10 mM dNTP mix
6 ïl of 25 mM MgCl2
1 ïl of DsRedprobe.fwdpr (10 pmol)
1 ïl of DsRedprobe.revpr (10 pmol)
0.25 ïl of AmpliTaq Gold polymerase
2 ïl of diluted genomic DNA in TE were added to 17 ïl of the master mix and the tubes were kept on ice. The thermal cycler GeneAmp PCR System 2400 was programmed appropriately (as presented in the Table below) and then the PCR cycle was repeated 35 times.
Controls for the PCR reaction were prepared in the same manner as the genomic DNA sample and they included:
blank- 2 ïl of water- negative control
non-transformant Rut-C30 2 ïl- negative control
PHEN54RQDsR plasmid 2ïl- positive control
DsRed original 2ïl -positive control
The PCR product was visualised with the aid of a 2% agarose gel. The gel was prepared (melted and cooled to approximately 60oC) and the GelRed in the amount of 8 ïl/200ml was added. Next, 10 ïl of the PCR product was placed in a fresh Eppendorf tube and 1.5 ïl of loading buffer (6 x) were added. After a brief spin, the mixture was loaded onto the gel and the reaction was run at 110 V.
The photograph of the gel was obtained.
Selection of mutant strains
The first control plate -non- transformant T. reesei plated on PDA and hygromycin B, was observed not to have any colonies growing on it.
The colonies from the mutant T.reesei strain transformed with the pHEN54RQDsRed DNA plasmid were counted after the first round of selection - the total number of transformants was 2. The transformation efficiency was therefore calculated to be 1 transformant per ïg of DNA (Transformation efficiency= number of transformants/ amount in ïg of the pHEN54RQDsRed DNA added). Only large, stable colonies presenting dynamic growth were counted as transformant colonies. The negative control plate (where DNA was not precipitated onto tungsten beads) was found to have no colonies growing.
Only colonies that grew on the plate after the second round of selection ( PDA + 60U/ml hygromycin B) and those growing on the plate covered with cellophane were regarded as true transformants and further used in the experiment.
Quality of DNA.
The purity and concentration of DNA was tested using a spectrophotometer. The readings at dparticular wavelengths were:
260 nm: 0.602
230 nm: 0.631
280 nm: 0.338
320 nm: 0.080
The purity was established by calculating the ratio of absorbance at 260 nm to the absorbance at 280 nm (O.D 260/280) and was found to be 1.78 whereas the reading at O.D 260/230 was 0.95. The concentration of non-diluted DNA was calculated to be 3 ïg/ïl.
The agarose gel electrophoresis was performed to further investigate the quality of the chromosomal DNA and the photography of the gel is presented in Figure 1. The sample of interest can be seen in the lane 8 (Group 1)
PCR product visualisation
The PCR product was visualised on the agarose gel under the UV illumination and a picture of the gel was taken (Fig. 2)
As mentioned before, assessment of gDNA purity was carried out using two methods. First of them was optical density (absorbance) where the measurements are based on the Beer-Lambert Law that identifies the relationship between the concentration of DNA and absorbance. The A260/A280 ratio for our genomic DNA sample was calculated to be 1.78, which indicated very pure DNA free from protein contamination- since rations 1.7-2.0 are generally accepted as those representing a good-quality samples (with 1.8 ratio being described as most desirable). However, this method in many research papers was found to suffer from some significant limitations 16,17; for example, it does not tell if the DNA is degraded. Therefore the second method used to assess the quality and purity of our sample was agarose gel electrophoresis, that was performed to address issues related to optical density technique and confirm (or question) our spectrophotometer readings.
As it can be seen in the photography of the gel (Fig. 1) the lane representing our sample (lane 8) contains some bright, smeared DNA bands. This result is nearly completely opposite to what we were expecting to see (especially after taking the quality readings from the spectrophotometer)- we expected gDNA present on the gel in the form of one relatively large-size band. Admittedly, there is a band visible on the lane, but the excessive smearing suggests DNA degradation (perhaps some nuclease contamination). The other possible explanation is that too much DNA was accidentally loaded onto the gel and hence the observed result. Surprisingly, the control genomic DNA from the non-transformant strain (lane 12) produced nearly identical results on the gel as the sample of interest (transformant). The non-transformant DNA also seems to be degraded and it also produced one band on the gel of the same size as the transformant strains. Since the control sample has less DNA in it than the transformant samples it should rather be visible on the gel in the form of a band that is located lower (smaller size) on the gel than the band of the transformants- as they have more DNA in form of the inserted plasmid.
The agarose gel image visualising the PCR product (Fig. 2) clearly indicates that transforming plasmid pHEN54RQDSsRed was integrated into the host organism and successfully amplified -the band at 250 kb (exactly as expected) can be taken as evidence for that integration. All of the transformant genomic DNA samples can be seen to give the same sized band (250 bp) except for the sample in the lane 18 whose DNA concentration was evidently too high and the sample had to be diluted and reloaded (reloaded into lane 16). Our sample of interest (lane 19) gave a visible band at 250 bp. However, the band is not as exposed and clear as in other samples because of the primer-dimers formation (large sized bright blob present below the band). Unexpectedly, the control Rut-C30 (non-transformant) in lane 5 was found to have an amplification product (250bp) even though we should not see any DsRed there since that control was not subjected to transformation. The most probably explanation of this is a human error when handling samples- the strains of transformants and non-transformants must have been accidentally mixed after the secondary selection (the strain did not grow on the plate after the second round of selection). The rest of the controls that is water (negative control) and both DsRed plasmids (positive controls)- gave the expected results.
In conclusion, our experiment that aimed at genetic transformation of Trichoderma reesei by microprojectile bombardment was successful as all of our samples that were subjected to biolistic bombardment with DNA- coated tungsten particles were indeed found to be stable transformants.
What type of transformation markers are available for fungi?
Generally speaking, transformation markers available for fungi include: nutritional (complement an auxotrophix requirement) or selectable antibiotic-resistance markers.
Drug-resistance markers are most popular and there is quite a few of them that have proved to be suitable for this purpose. Some of the markers include: hygromycin B resistance; phleomycin resistance; bialophos resistance; sulfonylurea resistance; benomyl resistance; KalnaImycin, G418 resistance
Acetamdase (AmdS)- which actually is not an antibiotic resistance gene, it encodes an enzyme (acetamidase) that gives the ability of growing using just acetamide (as the only carbon source) 18
2. What is DsRed1-E5 and what is it used for?
DsRed1- E5 protein otherwise known as the Fluorescent Timer, is a mutant of DsRed1 protein. The protein has an increased fluorescence intensity thanks to two amino acid substitutions (Val105Ala and Ser197Tyr) and it also changes its colour as time passes- after synthesis it start off with green light emission but after few mutations of the GFP-like fluorophore, it shifts to longer wavelengths and starts emitting red light (this usually means that the protein reached its mature stage).It has been established that the rate of colour changing is not correlated to protein concentration and thus can be used to trace expression that is time dependent.15 The DsRed1-E5 is used in fluorescent microscopy ('ready' tag) The unique property of the protein can be utilised in observations of for example cell differentiation (on/off gene expression).
3.what is the difference between genetically encoded and chemical fluorescent markers?