Flashcards in Semester 2 Deck (43):
Stages of a gene cloning experiment
1. Prepping the linear molecules that are to be joined, usually a vector and an insert.
2. Joining the DNA fragments (ligation).
-Annealed cohesive ends can be joined covalently by DA ligase, forming a phosphodiester bond between a 5' phosphate and a 3' hydroxyl. ATP is required.
3. Introducing the ligated molecules into a host organism (transformation).
-The process of uptake of naked DNA into cells, called transformation, requires treating E. coli cells to render them permeable to DNA (competent)
Preparation of competent cells: E. coli are rendered competent by chemical means by being washed into buffers (TFB1 and TFB2), which induce a transient permeabilisation of the outer membrane.
Transformation by heat shock: By exposing cells to a sudden increase in temperature, a pressure difference between the outside and the inside of the cell is created, inducing the formation of pores. At normal temperature, the cell walls self-heal.
Use of restriction enzymes
Restriction enzyme linearises the plasmid, producing sticky ends that can be ligated.
Use of DNA ligase
DNA ligase joins together sticky ends with a phosphodiester linkage, which recircularises the plasmid.
Use of calcium chloride
Calcium chloride gives Ca2+ ions which induce transient permeabilisation of the bacterial outer membrane and reduce electrostatic repulsion between the lipopolysaccharide layer and DNA which are both negatively charged.
Colonies / weight of DNA
Use of KCl (in PCR)
KCl neutralises charge present on the DNA backbone.
Use of MgCl2 (in PCR)
MgCl2 increases specificity and efficiency as a divalent cation.
Use of Tris-HCl (in PCR)
Tris-HCl acts as a buffer to keep a narrow pH range.
Use of dNTP mix (in PCR)
dNTP mix adds to the DNA sequence past the primer.
Temperatures: 94˚C, 50˚C, 72˚C.
In DNA profiling by Restriction Fragment Length Polymorphism (RFLP) analysis, DNA is digested using restriction enzymes and then separated using electrophoresis.
DNA fingerprinting analyses polymorphisms associated with short DNA repeats scattered across the genome called minisatellites.
Size exclusion chromatography
A procedure in which molecules can be fractioned according to size as they pass through a column packed with a gel matrix. As the proteins flow past the porous beads, they can diffuse into the internal pore structure of the beads to an extent that depends on their size.
Smaller molecules diffuse further into the pores of the beads and therefore move through the column more slowly, while larger molecules enter only a fraction of the pores and thus move through the column more quickly.
Molecules that are too big to enter the pores at all are excluded from the beads, and so can only pass through the volume of solution surrounding the beads in the column; these molecules therefore elute from the column first.
Practical uses of gel filtration
Desalting or buffer exchange
Purification of macromolecules
Analysis of the oligomeric state of proteins and protein complexes.
Ion exchange chromatography
This method separates two proteins of interest based on differences between surface charge as a result of ionisable amino acid side groups.
Cation exchanger: Negatively charged matrix that binds positively charged proteins.
Anion exchanger: Positively charged matrix that binds negatively charged proteins.
PI = the pH at which at which a given protein has no net charge.
-Solution pH > pI = -ve
-Solution pH < pI = +ve
Eluting proteins from the matrix in ion exchange chromatography
Change the pH of the solution to change the proteins charge using a buffer, by causing protonation/deprotonation.
Add salt to the buffer, salt ions (Na+, Cl- or PO42-) compete with the proteins for binding to the resin.
A less versatile method but can be more efficient.
A specific ligand is present on the beads and will recognise a specific structure in the protein of interest and bind to it.
Histidine likes to bind to nickel
-It is possible to engineer a protein that has a his-tag (chain of 6 His amino acids). The protein will bind to the nickel ions on the beads and will not pass through the column.
-Proteins without this engineered His-tag will pass through the column.
To release the proteins, imadazole is added which competes with the histidine to bind to the nickel.
Harden and Young found that...
Organic phosphate esters were being produced due to inorganic phosphate levels decreasing
Glycolysis requires low molecular weight cofactors
Lactose dehydrogenase and fermentation
Lactose dehydrogenase generates pyruvate, NADH and H+ from lactose and NAD+. The equilibrium of this reaction lies far to the left. Therefore, to generate pyruvate and NADH, the products have to be removed.
Protons are removed using an alkaline reaction medium and the pyruvate is reacted with hydrazine to form a hydrazone. The formation of the remaining product, NADH, is measured by its absorbance at 340 nm.
Use of EDTA (in anaerobic respiration)
EDTA is also included in the buffer to remove any divalent metal ions in the solutions
Use of MgCl2 (in anaerobic respiration)
MgCl2 is a cofactor essential for the enzymes involved in glycolysis. Stabilises ATP, cancels out charge
Use of apyrase (in anaerobic respiration)
Apyrase solution is used to prevent any ATPase or ADPase activity
DCPIP is a dye that makes it possible to measure the respiratory activity of a mitochondrial preparation with a spectrophotometer
Cyanide blocks cytochrome oxidase (complex IV) and prevents coupled and uncoupled respiration with all substrates
An uncoupler would inhibit respiratory electron transport in the mitochondria because electron transfer is coupled to proton transfer across the membrane so is one of these processes stops, the other stops too
Ways of measuring respiratory activity
One way of measuring this respiratory activity would be to measure the uptake of oxygen.
An alternative which is experimentally convenient and sensitive is to use a different acceptor in place of oxygen.
-Various dyes are able to tap off the reducing equivalents from the respiratory chain. An example is 2,6-dichlorophenol indophenol (DCPIP), which is deep blue in the oxidised form but colourless when reduced.
A useful indication of which compounds are thermodynamically capable of reducing or oxidising which other compounds is given by their oxidation-reduction potentials. Compounds at a low potential are oxidised by compounds at a higher potential
Malonate inhibits the oxidation of succinate to fumarate in the Citric Acid cycle of respiration. The electrons that would have oxidised the succinate are transferred to the DCPIP acceptor, resulting in a rapid change of colour.
Malonate is a competitive inhibitor
Investigating fermentation in yeast
Fermentation is an anaerobic cellular process in which organic compounds are converted into simpler molecules, and chemical energy is produced
For every molecule of pyruvate that is fermented, one molecule of CO2, one of ethanol, and one of NAD+ are generated (Remember, each glucose molecule that enters glycolysis yields two molecules of pyruvate).
We can measure the quantity of CO2 generated by measuring the amount of liquid the gas displaces from a fermentation reaction in a fixed volume
0.6M Tris.0.4M lysine-HCl pH 9.7 binds to acetaldehyde and removes it from the equilibrium
S.cerevisiae is not able to use lactose as a substrate
The fermentation reaction in yeast
The fermentation reaction (pyruvate --> acetaldehyde --> ethanol) catalysed by alcohol dehydrogenase (ADH) is reversible; incubation of ethanol with ADH can generate NADH and acetaldehyde in a stoichiometric fashion.
Therefore by incubating the products of a fermentation reaction with excess ADH we can drive the reaction backwards, and by measuring the NADH we can calculate the amount of ethanol that was generated by the fermentation.
Main products synthesised by Dunaliella
Main products that can be synthesised from Dunaliella:
β-carotene is more successful as a commercial product because the cells produce the product quickly and in large amounts and because it can be produced by cultures that are low in cost and extremely effective.
β-carotene is also used in food colouring, anti-cancer drugs, vegetable oils and in aquaculture
Dunaliella is a halotolerant algae that uses glycerol as a compatible solute to protect enzymes from inactivation and inhibition due to high salinities.
If the external salinity is increased, water flows from the cells; this triggers the synthesis of glycerol either by degradation of the storage product starch or by incorporation of photosynthetic CO2 fixation products into glycerol. The production of glycerol allows the cells to regain water from the medium and to return to their original volume
The effect of increasing osmotic pressure on microbial cells depends on whether they possess a cell wall. If they do, an increase in external osmotic pressure causes a decrease in turgor pressure as water flows from the cells. In wall-less cells, an increase in extracellular osmotic pressure leads directly to a decrease in cell volume which is detrimental to growth.
YsxC protein is an essential protein found in E. coli which means the gene is essential in survival. Its sequence suggests that it belongs to a class of proteins that bind GTP and is involved in the assembly of ribosomes
If we could find the 3d structure of this protein then we could synthesise a compound that fit into the active site of the protein, blocking it.
In order to do this however we need significant quantities of the protein in a pure form. The best way to do this is using a host such as E.coli.
Recombinant proteins are used for...
Targeted drug design
Structural and functional studies
Immunogen for antibody generation
Limitations of using bacteria as a host
Lack of post-translational modifications
Folding and solubility can be an issue
Its purpose is for protein sample preparation.
Use of mercaptoethanol (in Laemlli buffer)
Mercaptoethanol reduces sulphide bonds of the proteins to allow proper separation not by shape but by size.
Use of SDS (in Laemlli buffer)
SDS binds to all the proteins positive charges giving overall negative charge, while also denaturing proteins and subunits to help separation.
Sodium dodecyl sulphate (SDS) is an anionic detergent applied to protein samples to linearize proteins and give them negative charges
Use of bromophenol blue (in Laemlli buffer)
Use of glycerol (in Laemlli buffer)
Increases density of the sample.
Use of polyacrylamide gel (in protein separation)
The pores of an agarose gel are too large to sieve and separate out proteins based on their size. Therefore, polyacrylamide gel is used for proteins as the pores are smaller