Genome editing in diatoms Flashcards
What is a diatom?
Diatoms are single-celled algae. They are the only organism on the planet with cell walls composed of transparent, opaline silica.
Diatoms have light-absorbing molecules (chlorophylls a and c) that collect energy from the sun and turn it into chemical energy through photosynthesis.
Diatoms produce 20-30% of the air we breathe. Through carbon fixation, they remove CO2 from the atmosphere and convert it to organic carbon in the form of sugar, and O2 is released in the process.
Diatoms produce long-chain fatty acids. They are an important source of these energy rich molecules that are food for the entire food web.
Diatoms are the most diverse protists on earth.
Diatoms are particular about the quality of water in which they live. For example, species have distinct ranges of pH and salinity where they will grow. Diatoms also have ranges and tolerances for other environmental variables, including nutrient concentration, suspended sediments, flow regime, elevation, and for different types of human disturbance. As a result, diatoms are vital for assessment and monitoring biotic conditions of waters.
What is electrophoresis?
Electrophoresis is the movement of charged particles in a fluid or gel under the influence of an electric field
Why do we wash cells with sorbitol?
Sorbitol is an osmotically active sugar alcohol, and washing homogenate with sorbitol before cell lysis has been shown to significantly improve the purity of DNA extractions. Sorbitol does not pass cell membranes and likely acts by drawing the cytosol out of the cell. Therefore polyphenols and polycsaccharids would be removed, and the DNA will remain.
What is a vector construct?
A vector is typically a plasmid which can inject genetic materials into cells. The vector needs to have a specific nucleotide sequence.
A construct is a vector which has been modified to include the genetic material which is wanted to inject into the cell.
What is the PAM-target site?
“Protospacer adjacent motif” (PAM) is a short DNA sequence - typically 2-6 bp - which follows the DNA region which is target for cleavage by the CRISPR system, like CRISPR-Cas9. PAM is neccessary for the Cas nuclease to be able to cleave, and is normally found 3-4 nucleotides downstream of the cleavage site.
Another critical function of PAM is that the Cas nuclease will search for it before it dissolves the viral DNA to cleave it. When Cas identifies the correct PAM, it will check is the upstream region matches with the guide RNA before it edits.
What is biolistic transformation?
The approach of biolistic transformation requires specific technical tools: naked DNA er bound to gold or tungsten microparticles og is shoot into the living plant tissue by a particle gun. A certain number of cells will survive the bombardment and will integrate the original DNA into their own genome by illegitimate recombination and express the incorporated genetic information.
The most popular set-up for a particle gun consist of a vacuum chamber where DNA-coated particles accelerates down through the gun barrel with the plant tissue by helium overpressure.
How is screening for gene targeted mutations performed? (7 steps)
- A genetic report with the mutation that is being tested for is delivered.
- Blood test is sent to First Genomix
- DNA extraction
- DNA amplification and -sequencing
- Analysis of the results
- Interpretation of the genetic results in the report
- Genetic counseling is offered
How does the high blue light experiment works?
Chemiluminescent objects use chemical energy to produce light. The basic principle behind the reaction is that the reaction between the chemicals involved releases enough energy to excite the electrons in one of the reaction partners from the ground state to the excited state. This causes the electrons to “jump” to a higher energy level, then fall back and release light (energy). Depending on the chemicals involved, many different colors can be produced.
What is transfection?
Transfection is a process in which you can artificially introduce nucleic acids (DNA or RNA) into cells by using means other than viral infection. Such introductions of foreign nucleic acids by different chemical, biological or physical methods can result in a change of the cells properties, which can let us study gene function and protein expression i the cells context.
What is electroporation?
Electroporation is a very efficient method for transfection.
How is electroporation applied?
During electroporation, an electric pulse is used to made temporally pores in the cell membrane in which the nucleic acids can pass.
What is the process of electroporation?
Host cells and selected cells are suspended in a conductive solution and an electric circuit is closed around the mixture. An electric pulse with optimized charge and which only lasts for a few mikroseconds to one milliseconds is let through the cell suspension. This disturbs the phospholipid bilayer in the membrane which results in the production of temporarily pores. The electric potential across the membrane is elevated at the same time to let charged molecules like DNA across the membrane through the pores in a way that is similar to electrophoresis.
What is electrophoresis?
Electrophoresis is the movement of charged particles in a fluid or a gel under the affect of an electric field.
What is the four basic steps for electroporation?
(1) Tp prepare cells: prepare the cells by suspending them in a electroporation buffer.
(2) To apply an electric pulse: apply an electric pulse to the cells in the presence of specialized buffers and nucleic acids. The electric pulse produce a difference in the potential across the cell membrane and induce temporally pores in the membrane.
(3) To return cells to growth condition: return the cells to appropriate growth conditions for them to recover.
(4) To analyse cells: analyse the cells for gene expression or silencing.
What is the purpose of cell isolation?
Cell isolation is a method for separating and transfer certain cells from a complex mixture of cells to get a few single cells or to sort the cells according to a chosen property and thereby generating a homogenous cell population.
