Results from the biolistic bombardment and incubation revealed no presence of transformants on any plate. The next step was subjecting the isolated the chromosomal DNA to gel electrophoresis (Fig.1) in a bid to determine the value of the isolated DNA. Figures on the Biophotometer revealed the DNA concentration to be 1331 ng/µL as well as the DNA volume for use as PCR which registered as 0.75 µL. To check for the integration of DsRed gene to the host genome the products from the PCR were subjected to agarose gel electrophoresis (Fig.2).
The gene used as a transformant for Trichoderma reesei was DsRed gene. The process of bombardment was followed by testing the plates for signs of transformants through incubation. The results indicated no signs of presence of transformants. The possible causes for the absence are:
The DNA particles failed to hold on to the gold particles. There was a likelihood of the spores being destroyed due the force from the gene gun. If the age of the spemidine used in this test is approximately over 3 years old, it may lead to a hitch in the precipitation of the DNA. Another possible reason was failure of the Hygromycin gene to reach the spores. To ensure the experiment proceeded, the transformant was replaced with C5TH strain which was about one year old. Isolated chromosomal DNA’s quality was then checked by subjecting it to agarose gel electrophoresis. The image below (Figure 1) is a representation of band formations during the process of checking for the quality of the DNA as viewed under UV light. (Thieman & Palladino, 2009).
The DNA OF C5TH that i worked on is the one represented in the 5th well in the diagram. The results show the band as exhibiting longer branches and smear than all the other bands. The absorbence of DNA : protein is shown as 1.70, the DNA is therefore good in quality as it falls within the recommended range of 1.6-1.8. After isolation of the DNA, the DNA is then amplified by PCR which serves to indicate that the DsRed gene successfully combined with the host genome. PCR traces can be found spotted in the DsRed gene. Amplification is then followed by the agarose gel electrophoresis to enable clear visualization of the DNA bands. The figure below (Figure 2) depicts a representation of the presence of DsRed gene in the DNA.
Rut C30 which is plasmid DNA was used as the control and was included in the various wells. No bands were observed in the control which implies there is no DsRed gene present. The pDNA shows the band at 250 bp which means that pHEN54RQDsRed DNA which is itself a positive control. The plasmid DNA Rut C30 displays no band implying the absence of pHEN54RQDsRed DNA and for this reason it is said to act as a negative control. My sample well C5TH displays the band at 250bp indicating that pHEN54RQDsRed DNA is present.
The results indicate that pHEN54RQDsRed DNA transformed effectively in Trichoderma reesei which is a filamentous fungus. The process of isolation of the DNA was carried out including checking for its quality through agarose gel electrophoresis. The DNA concentration was registered at 1331 ng/µL. After amplification, the PCR product when checked under UV light indicated band of 250 bp.
There exists a variety in type of genes used as markers for fungi. One system is the use of drug resistance genes the commonly used being hph gene (hygromycin B resistance). This gene has been preferred owing to proven effectiveness in a majority of the cases. Another marker used to indicate transformation in fungi is auxotrophic markers such as pyrG. These are able to note presence or deficiency of certain components in the media. These are advantageous in that they allow for positive/negative gene selection thereby allowing the possibilities of carrying out several transformation tests (Weld et.al. 2006).
Fluorescent proteins are used as markers to trace cellular behaviour as well as for use in DNA tagging by use of multicolour imaging. DsRED-E5 is the recent make of fluorescent proteins and was designed to reform its colour over time from green to red regardless of the concentration of the genes. The prevalence ratio of the green to red in a DsRED-E5 marked sample can be used to trace the cell lineage as well as other cellular dynamics (Rossana, et.al 2004).
There exist different forms of fluorescent proteins; genetically encoded and chemical fluorescent markers. Genetically encoded fluorescent proteins refer to generating the fluorescent marker by way of inserting the protein dye into the genome and they use the prevalence of the colour to determine the cell characteristics. They are advantageous in that they can be tailor made to fit the intended purpose. Chemical fluorescent markers on the other hand use chemical combinations in the gene to determine the cellular dynamics (Thieman & Palladino, 2009).
Rossana, M.,Franken, C., Krogt, G., Bisseling, J., & Geurts, R. (2004). Use of the Fluorescent Timer DsRED-E5 as Reporter to Monitor Dynamics of Gene Activity in Plants. Plant Physiology. 35(135), 1879-1887.
Thieman, W. and Palladino, M. (2009). Introduction to Biotechnology, (2nd ed.) San Francisco, CA: Pearson Benjamin-Cummings Publishing Company.
Weld, R., Plummer, K., Carpenter, M.,& Ridgwa, H. (2006). Approaches to Functional Genomics in Filamentous Fungi. Web.