MSc Research Topic: Study of Molecular Mechanism for Pesticide Resistance in Spider Mites
Cloning of the target gene:
1) RNA extraction → check the concentration of RNA using spectrophotometer → cDNA (complementary DNA) synthesis →
2) DNA extraction →
1) and/or 2) → Amplification of the target gene by PCR (polymerase chain reaction) → gel electrophoresis (separate macromolecules based on size) → DNA ligation (to physically join the DNA vector to a gene of interest in order to form recombinant DNA, using an enzyme called DNA ligase) → transformation (to introduce recombinant DNA into host bacteria E. coli) → plasmid miniprep (use plasmid miniprep kit to extract plasmid DNA from E. coli cultures) → gel electrophoresis (to check the size of purified DNA) → sequence miniprep → sequence PCR → ethanol precipitation → use Hidi DNA polymerase →
3) Sequencing → data analysis
PhD Thesis: Complementary Approaches for Assessing Protein Sources in Fish Feed
The research of a nutritionally balanced, cost-effective, and environmentally & economically sustainable diet for farmed fish is becoming paramount. Researchers have studied the use of plant protein sources in the feed for different fishes. The main objective of my PhD research is to investigate the replacement of fishmeal with plant proteins in the feed for Finnish rainbow trout (Oncorhynchus mykiss) using stable isotope analysis which is a widely applied mathematical tool in ecological research. In this project, we have analysed the stable isotopes of carbon (C) and nitrogen (N) in different tissues (muscle, liver and fat), mucus and feces. The assimilated proportion of C and N was estimated using stable isotope mixing model which is a mathematical tool to estimate the proportion of stable isotopes in their mixture.
This kind of experiment with stable isotopes requires to keep the experimental fish on a diet containing single plant protein, but rainbow trout is a carnivorous fish and it is challenging to grow them on complete plant protein-based diet, especially juvenile rainbow trout. Therefore, the question is how do we overcome this difficulty? On the other hand, we also need to use protein sources with distinctive stable isotope ratio because the mathematical model will not function if the protein sources are similar in terms of stable isotope ratio. We have selected corn (C4 plant) and rapeseed (C3 plant), potato (C3 plant) and fishmeal in order to formulate and produce a diet with distinctive stable isotope ratio in terms of C and N. We have also tried to label the rapeseed and horse bean plants in the greenhouse by using heavy nitrogen fertilizer (ammonium nitrate containing 98% 15N) and heavy carbon dioxide (produced from the reaction between hydrochloric acid and sodium bicarbonate containing 98% 13C). However, we have failed to get sufficient seeds from the labelling experiment.
We have conducted two feeding experiments: 1st feeding trial (6 diets) & 2nd feeding trial (7 diets)
The main procedures followed:
Selection of protein sources based on the stable isotope ratio → diet formulation → diet production → set up experimental system → import juvenile fish from fish farm → start feeding trial after acclimation period → weighing and sampling of fish every week → sampling of mucus, tissues and feces from frozen fish → drying samples using freeze dryer → pulverizing freeze-dried samples → weighing (0.1-0.6 mg) of samples for stable isotope measurement → stable isotope measurement using elemental analyser interfaced with continuous-flow stable isotope ratio mass spectrometer → correction of stable isotope data → estimation of assimilated C and N incorporating stable isotope ratio of C and N into stable isotope mixing models → compare the results of stable isotope data with growth performance data (usually does not make sense) → whether it make sense or not, we need a further discussion → sometimes, the numbers show you some surprises → manuscript writing → polish the 1st draft of manuscript several times → submit...
Cloning of the target gene:
1) RNA extraction → check the concentration of RNA using spectrophotometer → cDNA (complementary DNA) synthesis →
2) DNA extraction →
1) and/or 2) → Amplification of the target gene by PCR (polymerase chain reaction) → gel electrophoresis (separate macromolecules based on size) → DNA ligation (to physically join the DNA vector to a gene of interest in order to form recombinant DNA, using an enzyme called DNA ligase) → transformation (to introduce recombinant DNA into host bacteria E. coli) → plasmid miniprep (use plasmid miniprep kit to extract plasmid DNA from E. coli cultures) → gel electrophoresis (to check the size of purified DNA) → sequence miniprep → sequence PCR → ethanol precipitation → use Hidi DNA polymerase →
3) Sequencing → data analysis
PhD Thesis: Complementary Approaches for Assessing Protein Sources in Fish Feed
The research of a nutritionally balanced, cost-effective, and environmentally & economically sustainable diet for farmed fish is becoming paramount. Researchers have studied the use of plant protein sources in the feed for different fishes. The main objective of my PhD research is to investigate the replacement of fishmeal with plant proteins in the feed for Finnish rainbow trout (Oncorhynchus mykiss) using stable isotope analysis which is a widely applied mathematical tool in ecological research. In this project, we have analysed the stable isotopes of carbon (C) and nitrogen (N) in different tissues (muscle, liver and fat), mucus and feces. The assimilated proportion of C and N was estimated using stable isotope mixing model which is a mathematical tool to estimate the proportion of stable isotopes in their mixture.
This kind of experiment with stable isotopes requires to keep the experimental fish on a diet containing single plant protein, but rainbow trout is a carnivorous fish and it is challenging to grow them on complete plant protein-based diet, especially juvenile rainbow trout. Therefore, the question is how do we overcome this difficulty? On the other hand, we also need to use protein sources with distinctive stable isotope ratio because the mathematical model will not function if the protein sources are similar in terms of stable isotope ratio. We have selected corn (C4 plant) and rapeseed (C3 plant), potato (C3 plant) and fishmeal in order to formulate and produce a diet with distinctive stable isotope ratio in terms of C and N. We have also tried to label the rapeseed and horse bean plants in the greenhouse by using heavy nitrogen fertilizer (ammonium nitrate containing 98% 15N) and heavy carbon dioxide (produced from the reaction between hydrochloric acid and sodium bicarbonate containing 98% 13C). However, we have failed to get sufficient seeds from the labelling experiment.
We have conducted two feeding experiments: 1st feeding trial (6 diets) & 2nd feeding trial (7 diets)
The main procedures followed:
Selection of protein sources based on the stable isotope ratio → diet formulation → diet production → set up experimental system → import juvenile fish from fish farm → start feeding trial after acclimation period → weighing and sampling of fish every week → sampling of mucus, tissues and feces from frozen fish → drying samples using freeze dryer → pulverizing freeze-dried samples → weighing (0.1-0.6 mg) of samples for stable isotope measurement → stable isotope measurement using elemental analyser interfaced with continuous-flow stable isotope ratio mass spectrometer → correction of stable isotope data → estimation of assimilated C and N incorporating stable isotope ratio of C and N into stable isotope mixing models → compare the results of stable isotope data with growth performance data (usually does not make sense) → whether it make sense or not, we need a further discussion → sometimes, the numbers show you some surprises → manuscript writing → polish the 1st draft of manuscript several times → submit...