fina Flashcards
Characteristics of a cloning vector
- Small in size
- Self replicating (ori)
- Restriction sites por endonucleases (MCS)
- Marker gene (like antibiontic resistance)
Characteristics of a expression vector
- Small in size
- Self replicating (ori)
- Restriction sites por endonucleases (MCS)
- Marker gene (like antibiontic resistance)
- Specific promoter and terminantor
Steps for cloning and transformating
- Cut plasmid and gene with restriction enzymes and pasting the gene in with ligases
- Insert the recombinant plasmid inside the bacteria (transfromaton)
- Plate the bacteria in solid growth media, using antibiotics
What is competent bacteria?
Bacteria that have incorporated foreing DNA. like a plasmid, from its sorroundings.
What is the transformation of a cell?
The process of genetic uptake by a competent cell
How is permeability induced in artificial competent cells?
Electric and chemical manipulation
What is SOC media?
Is microbial growth medium for the transfornation of competent cells, used as recovery medium and is added to the cell suspension for bacteria to recover at 37 ºC for 30 min.
It maximizes the efficiency of bacterial transformation
Why competent bacteria are sit in SOC media for 30 min at 37 ºC?
So bacteria repair their cell walls and start producing the antibiotic resistant protein.
What’s the best option when introducing a gene in a plasmid?
- cutting the plasmid with one Restriction enzyme
- Cutting it with two restriction enzymes
Cutting the plasmid with two restriction enzymes, so it doesn’t close back up without the gene.
What the right direction for the inserted gene to be, starting from the promoter?
5’ to 3’
What chemical induces transcription after you’ve isolated and put the plasmid-bearing bacteria to grow?
IPTG (Isopropyl β-d-1-thiogalactopyranoside)
Whats the lab method used for purifying proteins from a culture?
Affinity chromatography
How affinity chromatography works for protein prurification?
The cell macromolecules are put in a column with beads that have antibodies specific for the desired protein, which traps the protein and the rest of the molecules are washed away.
What is colony PCR?
A rapid PCR method to determine the presence of absence of the inserted DNA into the plasmid in a colony.
What GFP mean and from what species was first extracted?
Green Fluorescent protein, extracted from Jellyfish
How’s it used in molecular biology?
As a reporter gene that shows if a promoter is working inside a plasmid
If you add GFP to your bacteria and they glow green when seen under UV light, that means that the inserted gene is working well
True or false
False. It means that the promoter ins working well, but the gene may be inserted in the wrong direction
What procedure is needed to confirm that the gene inserted in a plasmid has the right orientation?
Colony PCR
What PAGE mean?
Plyacrilamide Gel Electrophoresis
Depending on what are the proteins separated in PAGE?
They separate depending on their molecular size
Why is agarose gel not used in PAGE?
Because proteins are too small to be separated well in an agarose gel.
A protein of 50 kDa is similar to a length of 75 - 10 bp
A protein of 250 kDa is equivalent to 500 bp
Hazards of acrylamide
Is carcinogenic (can cause skin cancer) and neurotoxic.
Acrylamide is unsoluble in water
True or false
False. It is soluble in water
What are the two main components of a polyacrylamide gel?
Acrylamide and bisacrylamide
Forms the cross links of linear polymers in a polyacrylamide gel
Bisacrylamide
Forms the lineat polymers in a polyacrylamide gel.
Acrylmide
Why acryamides reduces its toxicity and why?
Because it is formed and cross-linked matrix and the highly reactive vinyl groups have been lost. It reduces its toxicity in 95 %
What is ammonium persulfate (APS) for?
Starts polymerization
Compound used for speeding up the polymerization process in a polyacrylamide gel.
TEMED (Tetramethyl-ethylene-diamine)
pH, polyacrylamide content and use of the stacking gel in a PAGE.
pH 6.8
Polyacrylamide content around 4 %
Used to concentrate and line up the proteins before separating them
pH, polyacrylamide content and use of the resolving gel in a PAGE.
pH 8.8
Polyacrylamide content around 15 %
Used to separate the proteins base on their molecular weight
Why SDS-PAGE is horizontal
Because it would be dufficult to mount the stacking gel and resolving gel and oxygen inhibits the polymerization process of the polyacrylamide gel, so sandwiching it is ideal, to avoid oxygen interaction.
What SDS mean and what is it used for?
Sodium Dodecyl Sulfate. It is a detergent and that denatures proteins and coats them with a net negative charge. So proteins can migrate towards the positive pole.
Content of the loading buffer in a SDS-PAGE
- Bromophenol blue (for tracking)
- 2-mercapthoethanol (denaturing)
- Glyerol (gives the right consistency to work the bufferr)
- SDS (denaturing and negative charge)
- Tris-HCl (gives the right pH)
Content of the running buffer in a SDS-PAGE
- Tris-HCl (maintains pH)
- Glycine (Helps in the proper separation of the protein)
- SDS (denaturation)
Common dye to visualize proteins in the gel
Coomassie blue. It is added and the washed wih a solvent
If the staining of a SDS-PAGE is not enough to visualize the preoteins what can you do?
Do a silver staining
What is a Dalton?
Is a unit used to express the molecular weight of proteins, eqiuvalent to an atomic mass unit.
How proteins are separated in native PAGE?
They sepatate according to their net charge and size/shape
What is electrophoretic migration?
When most proteins have a net negative charge in an alkaline running buffer. The higher the negative charge, the faster it will migrate.
When do you prefer to run a native PAGE?
When you want to separate the active protein.
What a re the two main differences between SDS-PAGE and native PAGE?
- In native PAGE protein move according to their charge and shape. And in SDS-PAGE proteins are separated by the mass.
- In native PAGE you can obtain active proteins-
Can you obtain active proteins from SDS-PAGE?
No
What is an antigen?
A molecule (protein, polysaccharide or peptide) that is ussually found in the surface of a pathoen and is recognized by an antibody.
Can antibody kill a pathogen?
No. They only call other cells, like killer T cells, that will detroy the pathogen.
What technique do you use when you want to detect a specific protein from a sample?
Western blot
Major steps of western blot
- Sepatation of protein throgh SDS-PAGE
- Transfer of proteins to a solid support (blotting)
- Using the target protein as an antigen to mark it with radioactive antibody
- Wahing, to reduce interference
- autoradiography
Two most used solid supports for western blot
Nitrocellulose and nylon
Exposing the membrane of a western blot to a sheet of x-ray film is called:
Autoradiography
Primary antibodies are radioactive?
Usually not. Because its very expensive
What’s the antigen to secondary antibodies in western blot?
A conserved region of the antibodies that are constant amongs a partciular class of antibodies, like human IgG and mouse IgM
What are the two dimensions of 2D electrophoresis?
- Separation based on the isoelectric point
2. Separation by molecular weight
What is the isolelectric point?
The pH at wich a protein has no net electrical charge or is electrically neutral. The protein becomes a zwitterion
What is a zwitterion?
A molecule with functional groups. which at least one of them in postive and one negative, and the net charge of the molecule is 0
What is isoelectric focusing IEF?
The separation of proteins by they’re pH in a gel. The proteins are trapped at their isoelectric point in the gel strip
What is x-ray crystalography?
A method that uses X-rays to determine the folding of the proteins. The diffraction created by a protein is analyzed to determine arrangement of the atoms.
What is a crystal?
A solid material which constituents are highly ordered. A way to obtain a crystal is by vapor diffusion.
Wavelength of x-rays
0.01 - 10 nm
What is diffraction?
The phenomenon that occurs when a wave hits an obstacle.
Steps of crystalography:
Crystal —> diffraction pattern —> electron density map —> atomic model
What is the electron density map?
positions of the atoms, angles, chemical bonds, and some other information in the crystal can be determined.
What is a primary database?
They have experimentally derived data, like sequences and a littel more information. Once they got an accession number, info is not changed. Ex. NCBI, protein data bank
What is a secondary database?
Comprise data derived from the results of analyzing primary data. Also called fully curated databases. Ex. Uniprot, Protein information resorces.
What is the E-value?
The number of expected hits that could be found
just by chance. The lower the better
What is the query?
The sequence you introduce
Wht is the query coverage?
Is the percentage of your sequence aligned to a sequence in the Genbank to get the best match.
What is a phylogenetic tree?
a diagram that provides us information about the evolutionary relationship between organisms.
Hypothesis not facts.
What is the PDB archive?
Repository of atomic coordinates and other information describing proteins and other important biological macromolecules.
What is the world’s most comprhensive catalog of information about proteins?
UniProt
What is homology modeling?
obtains the three-dimensional structure of
a target protein based on the similarity between template (subject) and target sequences (query), computationally..
