Molekylär bioteknologi Flashcards
(88 cards)
1
Q
Nucleoside
A
(deoxy)ribose + nitrogenous base
2
Q
Nucleotide
A
Nucleoside + phosphate group
3
Q
Pyrimidine
A
Cytosine, Thymine, Uracil
4
Q
Purines
A
Adenine, Guanine
5
Q
Synthesize orientation
A
Moves from 3’ to 5’ to build a new strand in 5’ to 3’
6
Q
tRNA, rRNA, mRNA
A
Transfer RNA, translates RNA sequence to proteins using its anticodon.
Ribosomal RNA, binds to RBS and reads through RNA by leading tRNA in.
Messenger RNA.
7
Q
Protein read orientation
A
N-terminal to C-terminal
8
Q
How to isolate DNA
A
- Lyse cells with SDS+ NaOH + NaAc (do not expose for too long)
- Centrifuge and take supernatant
- Precipitate DNA, 3 parts ethanol 1 part isopropanol.
- Dissolve in Tris-HCl
- Include RNAse treatment in either step 1 or after step 3.
9
Q
DNA Absorbance wavelength
A
260 nm
10
Q
DNA labeling methods
A
- Fluorescent molecule
- Radiation: Phosphor Isotope
- Biotin/Digoxigenin
11
Q
What are the temperature needed for PCR reactions?
A
Denaturation: > 94C
Annealing: 50-60 C
Elongation through DNA polymerase: 68-72 C
12
Q
How many cycles are needed to get PCR strand?
A
3 cycles.
13
Q
3’ overhang
A
Horizontal cut closer to 3’ ends
14
Q
5’ overhang
A
Horizontal cut closer to 5’ ends
15
Q
Types of restriction enzymes
A
Type 2: Recognition site is restriction site.
Type 2S: Restriction site and recognition sites are different.
16
Q
Shuttle plasmid
A
Plasmid that works in different types of organisms, like E.coli and yeast. Needs however a second ori to function.
17
Q
Plasmid ligation ratio
A
1 part backbone 3 parts insert
18
Q
Possible vector systems
A
Plasmid for E.coli or Bacterial artificial chromosome.
Yeast artificial chromosome for yeast.
19
Q
Methods of cell transformation
A
- calcium chloride/heat shock:
Incubate on ice in Calcium ion solution then put in 42 C water. - electroporation:
Apply high voltage to create hole in membrane. Fast,easy and highly efficient but DNA needs to be very clean.
20
Q
Recombinant plasmid
A
Insert + vector plasmid
21
Q
Characteristics of an optimal expression vector
A
- high copy number
- selection marker
- inducible promoters
- strong RBS
- ATG 8 bp downstream of RBS
- effective terminator sequence
- optimized codon usage
22
Q
Lox P sites
A
Bp palindrome on both sides (13 bp each) and 8 bp in middle (GCATACAT). If two Lox P sites are in same direction Cre-recombinase can cut out one Lox p with the gene that is between the two Lox p. If Lox p are not in same direction, Cre-recombinase changes the orientation of middle genes.
23
Q
Southern Blotting
A
DNA from SDS-PAGE is transferred to nitrocellulose paper.
24
Q
Northern Blotting
A
RNA from SDS-PAGE is transferred to nitrocellulose paper.
25
Western Blotting
Protein from Native-PAGE is transferred to nitrocellulose paper.
26
TALEN
Transcription activator like effector nucleases
27
CRISPR
Clustered Regularly Interspaced Short Palindromic Repeats
28
ddNTP
dideoxynucleotide tri phosphate
29
Metagenomics
Study of all genomes within a specific environment
30
Ortholog
Similar protein in different species and have same function
31
Paralog
Similar protein in different species but have different functions
32
RNA composition in cells
2-5% mRNA
80-85% rRNA
10-12% tRNA
33
monocistronic mRNA
mRNA that codes for 1 protein
34
polycistronic mRNA
mRNA that codes for several protein
35
5' cap
Tri phosphate + Ribose + 7-methyl-guanosine
36
3' poly A tail
Adenosine monophosphate (Adenine base)
37
Spliceosome
Cuts out introns
38
Translation efficiency
Ribo-seq levels/ RNA-seq levels
39
Dicer
RNA endonuclease, produces siRNA (small interference RNA)
40
RISC
(RNA induced silencing complex) Protein that unwinds siRNA and uses antisense RNA to find complementary mRNA to degrade it. (Argonaute is a part of RISC complex)
41
Isoelectric point
pH where net charge is neutral for protein.
42
Proteom
Alla proteiner som genomet kodar för.
43
Homolog produktion
Produceras i värd "naturligt"
44
Heterolog produktion
Produceras i värd på grund av transformering, t.ex p.g.a expression vektorer.
45
UV våglängd för peptidbindningar
230 nm
46
UV våglängd för aromatiska amino syror
280 nm
47
Apo-structure
Enzyme without ligand
48
Holo-structure
Enzyme with ligand
49
Processive enzyme
Enzyme that is both endo/exo-acting
50
Induced pluripotent stemcells
Differentiated cells that have been reset into pluripotent stem cells.
51
Stärkelse
Amylose + amylopectin (maltos/isomaltos aka 2 glukos)
52
Alpha-amylas
Endo-acting enzym som klyver alpha-glukankedjor av minst 3 monosackarider.
53
glukoamylas
Exo-acting enzym som klyver alpha-glukankedjor från änden.
54
CBH
Cellobiohydrolas. Exo-acting. Aktivt säte är en tunnel. Bryter ned cellulosa till cellobios.
55
EG
Endo-glukanas. Klyver amorfa regioner av cellulosan.
56
BG
Beta-glukosidas. Bryter ned cellobios till glukos. Oftast brist på. Minskar effekt av produkt inhibering.
57
LPMO
Lytiskt polysackarid-monooxygenas. Metallo-enzym, binder kopparatom som bildar reaktiva syreföreningar som reagerar med glykosidbindningarna. Affinitet för kristalina substrat.
58
CBM
Carbohydrate binding module. Länkade via flexibla linkers till den katalytiska modulen.
59
FISH
fluorescens in situ hybridization
60
FPLC
fast protein liquid chromatography
61
IMAC
Immobilized metal affinity chromatography
62
GST-tag
Gluathione S-transferase tag
63
ELISA
Enzyme-linked immuosorbent assay
64
MALDI
matrix-assisted laser desorption/ionization
65
ESI
electrospray ionization
66
HPLC
High performance liquid chromatography
67
iTRAQ
Isobaric tag for relative and absolute quantitation
68
SILAC
Stable isotope labeling by amino acids in cell culture
69
Name and describe methods of modifying DNA
- Gibson Assembly
- Error-prone PCR
- Mutagenic two way primer
- Lox P site (use Cre-recombinase)
- Zinc finger nucleases (Fok1)
- transcription activator like effector nucleases (Fok1)
- CRISPR/Cas9
- CRISPRi/dCas9
- non-homologous end joining
- homology directed repair
- CRISPRa/CRISPRi
- change gene promoter
- increase copy number
- gene shuffling
- siRNA and RISC to cleave out DNA that create dsRNA
70
Name and describe methods of changing RNA and its translation efficiency (do not include DNA methods)
- Use of reverse transcriptase PCR to create new mRNA from DNA.
- antisense RNA
- change RBS to further away from translation start codon
- change Kozaq sequence
- Include Riboswitch in mRNA or loose to inhibit ribosome
71
Name and describe different methods of DNA sequencing
- Sanger sequencing (use of ddNTP)
- shotgun method (primer walking when encountering gaps)
- Pyrosequencing
- Ion torrent
- Illumina sequencing
- Pacific Bioscience sequencing
- Oxford nanopore
- ribo-sequencing
72
Name two ways of localization a specific mRNA sequence
- In vivo mRNA localization using MS2 protein.
- In vivo mRNA localization using dCas9.
73
Ways of transforming cells
- Heat schock/ calcium chloride
- Electroporation
74
Name different types of chromatography
- FPLC (machine)
- IMAC
- Immunoaffinty chromatography
- GST-tage (glutation S-transferase)
- His-tag
- Ion exchange chromatography
- Hydrophobic interaction chromatography
- size exclusion chromatography
Remember that proteins have to be cut with trypsin. Predicting where it will cut = peptide-fingerprinting
75
Name different ways of analyzing proteins and how to find concentrations in sample
- Bradford method (find concentration by creating standard curve with bovine albumin serum)
- UV (spectroscopy Beer-Lambert's law)
- SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis,denatured)
- Native-PAGE
- ELISA, direct or sandwich
- chrystallography
- small-angle X-ray scattering
- NMR
- electronmicroscope
- 2D gel
76
Name different types of mass spectrometry and its parts.
- MALDI
- electrospray ionization
- quadropole (magnets directing the ions)
- mass analysator
- MS/MS (knock each oligopeptide into peptide by smashing them against noble gas.)
- can do HPLC before to separate unwanted proteins and lower the amount of peptides
77
Name ways of tracking proteins from different cell cultures in tandem mass spectrometry
- stable isotope labelled by amino acids in cell culture
- isobaric tag for relative and absolute quantitation (reporter + balance group = isobaric tag and added with a peptide reactive group)
78
What is substrate/product inhibition?
Substrate/product inhibits and keeps signal on even level.
79
What happens to V_max and K_m in competitive inhibition?
V_max doesn't change, K_m becomes higher
80
What happens to V_max and K_m in non-competitive inhibition?
V_max lowers and K_max doesn't change
81
What happens to V_max and K_m in uncompetitive inhibition
K_m and V_max lowers
82
Name different methods of introducing new DNA to somatic stem cells
Lipofection and pronuclear injection
83
Name different ways of modifying genes of higher organisms
- Plasmids (Ori must be compatible)
- non-self replicative DNA
- virus vectors like adeono, AAV and retro
- Use of truncated viruses and packaging cells
For plants specifically:
- Ti-plasmid/ Agrobacterium transformation
- can target chloroplast with HDR (originally from bacteria)
84
Name ways of analyzing metabolic pathways
- Metabolite fingerprinting/footprint
- flux analysis
85
How do you find new enzymes in nature?
- Analyze and sequence genome of organism that are of interest (use fylogeni or sequence similarity networks to find suitable organisms)
- Forward genetics = find phenotype by randomly mutation in gene.
- Reverse genetics = start with a known gene, alter/disrupt and investigate any phenotypic changes that occur
- Look at gene cluster to see if there are any enzymes of interest, specially viable for operons.
- Do module walking, find a known module sequence and find what enzyme it is linked to.
- Functional screening (test enzyme activity on a group of candidate enzymes)
86
Describe and name methods of modifying enzymes so that it has the desirable trait
Rational design (figure out what should be altered):
- crystallography
- NMR
then change with point mutation, example Asp/glut.
or use directed evolution, often a combination of both.
Direct evolution needs high throughput screening.
87
Name methods of enzyme immobilization
Immobilization can be direct/indirect
- ultrafiltration membrane
- hollow fiber devices (cylindrical micelles + silicate)
- cross-linking (often with glutaraldehyde)
- physical adsorption (carrier or matrix)
- ionic binding (carrier or matrix)
- metallic binding (carrier or matrix)
- covalent binding (carrier or matrix)
- gel entrapping (matrix)
- fiber entrapping (matrix, watch out for fouling)
- micro encapsulation (carrier)
88
What are the effects of immobilization of enzymes?
- K_m changes mostly, not so much V_max/K_cat.
- K_m can lower if opposite charge
- K_m increase if diffusion problems or konformational/active site alterations
- good for shielding against pH changes and temp changes
