Planning Experiments in Plant Molecular Biology
1. Introduction into a possible project aiming to study the structure function relation of a protein involved in plant development. General discussion of the flow chart of the whole project and each step of it and also the methods going to be used during this possible experiment.
2. Choosing and identification of the protein involved in the experiments of functional studies. Protein polyacrilamide gel electrophoresis methods, RP-HPLC and capillary electrophoresis as a choice and their advantages and disadvantages. Protein sequencing.
3. Degenerate primer design for RT-PCR. Poly A RNA extraction and purification from plant tissue. Detailed discussion of the reverse transcription as an enzyme reaction including the reaction conditions, different enzymes could be used and the optimisation.
4. What has to be considered during the preparation of a cDNA library. Checking and screening the library. Choosing the appropriate clone and its further characterisation before sequencing. Sequencing. General characterisation of all the available protein expression systems including advantages and disadvantages.
5-6. Detailed characterisation of the bacterial expression systems can be purchased. Examples of their application for different target proteins.
7. How to design oligonucleotides for cloning a gene into the expression vectors? Explaining all the problems has to be considered during this step. Discussion all the trouble may arise during the use of the PCR reaction and also the methods to be applied to avoid it.
8. Detailed discussion all the steps (digestion, ligation and transformation) necessary to clone the amplified DNA into the expression vector, including the problems could arise during the process and has to be considered to avoid them. Bacterial transformation techniques and their advantages and disadvantages.
9. Confirming the sequence of the amplified and cloned sequence. Double digestion of the DNA with restriction enzymes, sub cloning into the expression vector.
Characterisation of host bacterial cells for cloning and protein expression and the condition of their usage.
10. Expression of the protein in a heterologous system. Problems may arise during the expression. Discussion of the improvement of the expression; induction, time and temperature. Expression of toxic proteins.
11. Protein extraction from bacteria. Analysis of the protein function with or without purification. Purification of the fusion proteins using different column chromatography steps. Discussion of the advantages and disadvantages of the application of the fusion protein technique. Methods to remove the “tag” from the expressed protein.
12. Modification primary structure of the gene through site directed mutagenesis to study protein structure function relation. Detailed characterisation of the methods available. Functional studies of the expressed, purified proteins.
13- 14. Characterisation of other in vitro and in vivo protein expression systems (cell free small and large scale systems, baculovirus, yeast, etc).
Petter Laake et al.: Research in Medical and Biological Sciences, Academic Press, 2015