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Biologi > Molekylär bioteknologi > Flashcards

Molekylär bioteknologi Flashcards

1
Q

Nucleoside

A

(deoxy)ribose + nitrogenous base

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2
Q

Nucleotide

A

Nucleoside + phosphate group

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3
Q

Pyrimidine

A

Cytosine, Thymine, Uracil

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4
Q

Purines

A

Adenine, Guanine

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5
Q

Synthesize orientation

A

Moves from 3’ to 5’ to build a new strand in 5’ to 3’

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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.

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7
Q

Protein read orientation

A

N-terminal to C-terminal

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8
Q

How to isolate DNA

A
  1. Lyse cells with SDS+ NaOH + NaAc (do not expose for too long)
  2. Centrifuge and take supernatant
  3. Precipitate DNA, 3 parts ethanol 1 part isopropanol.
  4. Dissolve in Tris-HCl
  5. Include RNAse treatment in either step 1 or after step 3.
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9
Q

DNA Absorbance wavelength

A

260 nm

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10
Q

DNA labeling methods

A
  • Fluorescent molecule
  • Radiation: Phosphor Isotope
  • Biotin/Digoxigenin
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11
Q

What are the temperature needed for PCR reactions?

A

Denaturation: > 94C
Annealing: 50-60 C
Elongation through DNA polymerase: 68-72 C

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12
Q

How many cycles are needed to get PCR strand?

A

3 cycles.

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13
Q

3’ overhang

A

Horizontal cut closer to 3’ ends

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14
Q

5’ overhang

A

Horizontal cut closer to 5’ ends

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15
Q

Types of restriction enzymes

A

Type 2: Recognition site is restriction site.
Type 2S: Restriction site and recognition sites are different.

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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.

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17
Q

Plasmid ligation ratio

A

1 part backbone 3 parts insert

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18
Q

Possible vector systems

A

Plasmid for E.coli or Bacterial artificial chromosome.
Yeast artificial chromosome for yeast.

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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.
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20
Q

Recombinant plasmid

A

Insert + vector plasmid

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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
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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.

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23
Q

Southern Blotting

A

DNA from SDS-PAGE is transferred to nitrocellulose paper.

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24
Q

Northern Blotting

A

RNA from SDS-PAGE is transferred to nitrocellulose paper.

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25
Q

Western Blotting

A

Protein from Native-PAGE is transferred to nitrocellulose paper.

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26
Q

TALEN

A

Transcription activator like effector nucleases

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27
Q

CRISPR

A

Clustered Regularly Interspaced Short Palindromic Repeats

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28
Q

ddNTP

A

dideoxynucleotide tri phosphate

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29
Q

Metagenomics

A

Study of all genomes within a specific environment

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30
Q

Ortholog

A

Similar protein in different species and have same function

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31
Q

Paralog

A

Similar protein in different species but have different functions

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32
Q

RNA composition in cells

A

2-5% mRNA
80-85% rRNA
10-12% tRNA

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33
Q

monocistronic mRNA

A

mRNA that codes for 1 protein

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34
Q

polycistronic mRNA

A

mRNA that codes for several protein

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35
Q

5’ cap

A

Tri phosphate + Ribose + 7-methyl-guanosine

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36
Q

3’ poly A tail

A

Adenosine monophosphate (Adenine base)

37
Q

Spliceosome

A

Cuts out introns

38
Q

Translation efficiency

A

Ribo-seq levels/ RNA-seq levels

39
Q

Dicer

A

RNA endonuclease, produces siRNA (small interference RNA)

40
Q

RISC

A

(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
Q

Isoelectric point

A

pH where net charge is neutral for protein.

42
Q

Proteom

A

Alla proteiner som genomet kodar för.

43
Q

Homolog produktion

A

Produceras i värd “naturligt”

44
Q

Heterolog produktion

A

Produceras i värd på grund av transformering, t.ex p.g.a expression vektorer.

45
Q

UV våglängd för peptidbindningar

A

230 nm

46
Q

UV våglängd för aromatiska amino syror

A

280 nm

47
Q

Apo-structure

A

Enzyme without ligand

48
Q

Holo-structure

A

Enzyme with ligand

49
Q

Processive enzyme

A

Enzyme that is both endo/exo-acting

50
Q

Induced pluripotent stemcells

A

Differentiated cells that have been reset into pluripotent stem cells.

51
Q

Stärkelse

A

Amylose + amylopectin (maltos/isomaltos aka 2 glukos)

52
Q

Alpha-amylas

A

Endo-acting enzym som klyver alpha-glukankedjor av minst 3 monosackarider.

53
Q

glukoamylas

A

Exo-acting enzym som klyver alpha-glukankedjor från änden.

54
Q

CBH

A

Cellobiohydrolas. Exo-acting. Aktivt säte är en tunnel. Bryter ned cellulosa till cellobios.

55
Q

EG

A

Endo-glukanas. Klyver amorfa regioner av cellulosan.

56
Q

BG

A

Beta-glukosidas. Bryter ned cellobios till glukos. Oftast brist på. Minskar effekt av produkt inhibering.

57
Q

LPMO

A

Lytiskt polysackarid-monooxygenas. Metallo-enzym, binder kopparatom som bildar reaktiva syreföreningar som reagerar med glykosidbindningarna. Affinitet för kristalina substrat.

58
Q

CBM

A

Carbohydrate binding module. Länkade via flexibla linkers till den katalytiska modulen.

59
Q

FISH

A

fluorescens in situ hybridization

60
Q

FPLC

A

fast protein liquid chromatography

61
Q

IMAC

A

Immobilized metal affinity chromatography

62
Q

GST-tag

A

Gluathione S-transferase tag

63
Q

ELISA

A

Enzyme-linked immuosorbent assay

64
Q

MALDI

A

matrix-assisted laser desorption/ionization

65
Q

ESI

A

electrospray ionization

66
Q

HPLC

A

High performance liquid chromatography

67
Q

iTRAQ

A

Isobaric tag for relative and absolute quantitation

68
Q

SILAC

A

Stable isotope labeling by amino acids in cell culture

69
Q

Name and describe methods of modifying DNA

A
  • 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
Q

Name and describe methods of changing RNA and its translation efficiency (do not include DNA methods)

A
  • 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
Q

Name and describe different methods of DNA sequencing

A
  • 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
Q

Name two ways of localization a specific mRNA sequence

A
  • In vivo mRNA localization using MS2 protein.
  • In vivo mRNA localization using dCas9.
73
Q

Ways of transforming cells

A
  • Heat schock/ calcium chloride
  • Electroporation
74
Q

Name different types of chromatography

A
  • 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
Q

Name different ways of analyzing proteins and how to find concentrations in sample

A
  • 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
Q

Name different types of mass spectrometry and its parts.

A
  • 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
Q

Name ways of tracking proteins from different cell cultures in tandem mass spectrometry

A
  • 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
Q

What is substrate/product inhibition?

A

Substrate/product inhibits and keeps signal on even level.

79
Q

What happens to V_max and K_m in competitive inhibition?

A

V_max doesn’t change, K_m becomes higher

80
Q

What happens to V_max and K_m in non-competitive inhibition?

A

V_max lowers and K_max doesn’t change

81
Q

What happens to V_max and K_m in uncompetitive inhibition

A

K_m and V_max lowers

82
Q

Name different methods of introducing new DNA to somatic stem cells

A

Lipofection and pronuclear injection

83
Q

Name different ways of modifying genes of higher organisms

A
  • 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
Q

Name ways of analyzing metabolic pathways

A
  • Metabolite fingerprinting/footprint
  • flux analysis
85
Q

How do you find new enzymes in nature?

A
  • 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
Q

Describe and name methods of modifying enzymes so that it has the desirable trait

A

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
Q

Name methods of enzyme immobilization

A

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
Q

What are the effects of immobilization of enzymes?

A
  • 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

Biologi (3 decks)

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