Summary
Samenvatting Gentechnologie I deel II, BCBT
- Course
- Institution
samenvatting van 2de deel van gentechnologie deel II door prof. berckx (voor goedkopere prijs stuur via messenger)
[Show more]
Seller
Follow
jarnewinderickx
Reviews received
Content preview
SAMENVATTINGGENTECHNOLOGIE I:
DEEL II
[Ondertitel van document]
Abstract
[Trek de aandacht van uw lezer met een interessante samenvatting. Dit is
meestal een kort overzicht van het document.
Wanneer u uw inhoud wilt toevoegen, klikt u hier en begint u te typen.]
Jarne Winderickx
[E-mailadres]
,INHOUD
1. VECTOREN.................................................................................................3
1.1 INLEIDING..................................................................................................................... 3
1.1.1 Bereiding van het te kloneren DNA.....................................................................3
1.1.2 Invoeren van het DNA fragment in gekozen vector............................................3
1.1.3 Inbrengen in de gastheer (GH)...........................................................................3
1.2 VECTOREN.................................................................................................................... 4
1.2.1 Plasmiden........................................................................................................... 4
1.2.1.1 Het plasmide ColE1 als modelsysteem voor de latere ontwikkeling van
kloneringsvectoren................................................................................................................. 4
1.2.1.2 Plasmiden als kloneringsvectoren............................................................................... 5
1.2.1.3 Voorbeelden............................................................................................................... 5
1.2.1.4 Plasmide transformatie in bacteriën........................................................................... 6
Chemische behandeling..................................................................................................... 6
Electroporatie..................................................................................................................... 6
1.2.2 Enkelstrengige DNA bacteriofagen.....................................................................7
1.2.3 Faagmiden.......................................................................................................... 7
1.2.4 Faagvectoren afgeleid van bacteriofaag.............................................................7
1.2.5 Cosmiden............................................................................................................ 9
1.2.8 Intermezzo: verkorten van gDNA......................................................................10
1.2.9 Supervectoren: YACs en BACs...........................................................................10
1.2.10 Keuze van de vector.......................................................................................11
1.2.10.1 Expressievectoren.................................................................................................. 11
1.2.10.2 Constructie van een genomische bank...................................................................11
1.3 NIEUWE GENERATIE KLONERINGSMETHODEN.......................................................................11
1.3.1 Klassieke klonering........................................................................................... 11
1.3.2 TOPO klonering: moderne kloneringen.............................................................12
1.3.2.1 TOPO TA klonering.................................................................................................... 12
1.3.2.2 Zero blund TOPO klonering....................................................................................... 12
1.3.3 Gateway klonering............................................................................................12
1.3.4 Creator klonering.............................................................................................. 14
1.4 OEFENING: NAADLOOS KLONEREN EN GENFUSIE..................................................................14
2. DNA LABELEN........................................................................................... 15
2.1 RADIOACTIEVE MERKERS................................................................................................ 15
2.1.1 Detectie............................................................................................................ 16
2.2 NIET-RADIOACTIEVE MERKERS......................................................................................... 17
2.3 ENZYMATISCHE METHODES VOOR NUCLEÏNEZUUR LABELEN....................................................18
2.3.1 5’-eindstandige merking...................................................................................18
2.3.2 3’-eindstandige merking...................................................................................18
2.3.3 Inwendige merking...........................................................................................18
2.3.4 Merking RNA moleculen....................................................................................19
2.4 CHEMISCHE SYNTHESE VAN NUCLEÏNEZUREN / PRIMERS / OLIGONUCLEOTIDEN...........................19
2.5 CHEMISCHE OF FOTOMERKING VAN NUCLEÏNEZUREN............................................................20
3. TRANSFORMATIE VAN BACTERIËN EN SELECTIE VAN TRANSFORMANTEN......20
3.1 TRANSFORMATIE........................................................................................................... 21
3.2 SELECTIE MET BEHULP VAN SELECTIE-MERKERS...................................................................21
3.3 TRANSFORMATIE-EFFICIËNTIE.......................................................................................... 21
3.4 KOLONIEHYBRIDISATIE................................................................................................... 21
4. DNA SEQUENERINGS METHODES................................................................22
4.1 SANGER METHODE / DIDEOXY SEQUENERING......................................................................22
4.2 SEMI-AUTOMATED SEQUENERING.....................................................................................22
4.3 SEQUENEREN VAN COMPLETE GENOMEN: SHOTGUN SEQUENERING.........................................23
1
, 4.4 CAPILLAIRE ELEKTROFORESE........................................................................................... 24
4.5 BLAST / BASIC LOCAL ALIGNMENT SEARCH TOOL: OVERSLAAN..............................................25
4.6 NEXT GENERATION SEQUENCING / NTG SEQUENCING..........................................................25
4.6.1 Pyrosequencing................................................................................................26
4.6.2 Illumina sequencing..........................................................................................27
4.6.3 SOLID sequencing / sequencing by oligoligation and detection........................28
4.6.4 Enkelvoudige DNA molc sequenering / Helicos sequenering.............................29
4.6.5 SMRT sequenering............................................................................................30
4.6.6 Isolatie DNA van interesse................................................................................30
4.6.7 ChIP sequencing...............................................................................................31
4.6.8 Ion Torrent sequencing.....................................................................................31
5. GENEXPRESSIE.........................................................................................32
5.1 HETEROLOGE / RECOMBINANTE GENEXPRESSIE IN PROKARYOTEN............................................32
5.1.1 Induceerbare promotor voor genexpressie.......................................................32
5.1.1.1 Trp operon................................................................................................................ 33
5.1.1.2 Lac-operon............................................................................................................... 33
5.1.1.3 Tac-promotor: hybride Trp en lac promotor...............................................................34
5.1.1.4 pL promotor.............................................................................................................. 34
5.1.1.5 T7 gen 10 promotor.................................................................................................. 34
5.1.1.6 Regeling ProU operon / osmoregulatie......................................................................35
5.1.1.7 Laboschaal vs industrieel......................................................................................... 35
5.1.2 Fusie-eiwitten................................................................................................... 36
5.1.3 Faag-display..................................................................................................... 36
5.1.3.1 faag-display van AL-fragmenten............................................................................... 37
5.1.3.2 VHH AL / nanobodies / Heavy-chain only AL.............................................................40
5.1.4 Controle eiwitsynthese.....................................................................................40
5.1.4.1 Codon gebruik: aanpassen aan organisme...............................................................40
5.1.4.2 N-terminale AZ bepalen eiwitstabiliteit mee.............................................................40
5.1.4.3 Eiwitopvouwing bevorderen: S-S brug vorming.........................................................41
5.1.5 Verwijderen van ABR-genen..............................................................................41
5.2 VOORBEELDEN VAN RECOMBINANTE THERAPEUTISCHE EIWITTEN.............................................42
5.2.1 mens IFN-beta klonering en expressie..............................................................42
5.2.2 menselijk groeihormoon / MGH klonering.........................................................43
5.2.3 Biologische inperking van genetisch veranderde L. lactis.................................44
5.2.4 Recombinant influenzavaccin aangemaakt in E.coli.........................................45
2
, 1. VECTOREN
1.1 INLEIDING
Kloneren = invoeren DNA segment (uit bacterie, plant, dier,…) in geschikte
dragermolecule / vector om daarna stabiel te doen vermenigvuldigen in
gekozen GH
o Vector bepaald door GH + moet kunnen worden doorgegeven aan
volgende generaties
Stappen kloneringen: bereiden DNA-fragment invoeren in vector
inbrengen in GH zoeken naar GH die vector hebben opgenomen (/
positieve klonen) amplificeren
o Hoe amplificeren? klonaal uitgroeien (= alle bacteriën op
agarplaat doen groeien verdunnen isolatie 1 kolonie
monocultuur
1.1.1 BEREIDING VAN HET TE KLONEREN DNA
= nieuw DNA (PCR-product, DNA-fragmenten na restrictiedigest,…) als in
vivo geïsoleerd DNA (gDNA / genomisch DNA)
Ook onderscheid: klonering 1 enkel fragment of gehele mengsels
fragmenten
o Gehele mengsels: om cDNA en gDNA banken te maken
Hoe? cDNA’s van alle voorkomende mRNA in cel kloneren
1.1.2 INVOEREN VAN HET DNA FRAGMENT IN GEKOZEN VECTOR
Oorspronkelijk: ligatiereactie tussen insert + vector (= klassieke
klonering)
1.1.3 INBRENGEN IN DE GASTHEER (GH)
Transformatie vs transfectie: vreemd DNA binnenbrengen in virussen,
bacteriën,.. vs vreemd DNA binnenbrengen in eukaryote cellen
3
The benefits of buying summaries with Stuvia:
Guaranteed quality through customer reviews
Stuvia customers have reviewed more than 700,000 summaries. This how you know that you are buying the best documents.
Quick and easy check-out
You can quickly pay through credit card or Stuvia-credit for the summaries. There is no membership needed.
Focus on what matters
Your fellow students write the study notes themselves, which is why the documents are always reliable and up-to-date. This ensures you quickly get to the core!
Frequently asked questions
What do I get when I buy this document?
You get a PDF, available immediately after your purchase. The purchased document is accessible anytime, anywhere and indefinitely through your profile.
Satisfaction guarantee: how does it work?
Our satisfaction guarantee ensures that you always find a study document that suits you well. You fill out a form, and our customer service team takes care of the rest.
Who am I buying these notes from?
Stuvia is a marketplace, so you are not buying this document from us, but from seller jarnewinderickx. Stuvia facilitates payment to the seller.
Will I be stuck with a subscription?
No, you only buy these notes for $3.80. You're not tied to anything after your purchase.
Can Stuvia be trusted?
4.6 stars on Google & Trustpilot (+1000 reviews)
62890 documents were sold in the last 30 days
Founded in 2010, the go-to place to buy study notes for 14 years now