Vectors Flashcards
Cosmids
Plasmids with med/high copy number origin + lambda packaging sequence (Cos ends)
- 8kp
- introduced into E.coli by transduction
An example of a cosmid
SupercosI -> selection marker for eukaryotic cells with a SV40 origin
What are the benefits of using a cosmid?
Injects and circularises like phage DNA but replicates as a normal plasmid without phage functions
BACS
Plasmid with origin of F1 - plasmid with copy number 1
- contains genome inserts e.g. sacB replaced lacZ or insertion of transposon
- includes lambda cos N + PI loxP sites + restriction enzyme sites
- can be transferred efficiently without packaging
YACS
Plasmid with additional autonomously replicating sequence
-> generates 2 arms that ligate to genomic DNA -> linear construct
Benefit of YACs
No hard size limit, introduced by electroporation
YAC vs BAC
YAC’s are less stable and more labour-intensive so slower than BACs. Both can be manipulated by homoogous recombination but easier in YAC’s.
Construct design for high-level eukaryotic expression:
- Strong + constituive promoter
- Intron included
- PolyA signal -> defined 3’ end to mRNA + expor mRNA
- Remove unnecessary untranslated sequences (au reduce stability)
- Optimisation of transgene for translational efficiency e.g. AUG in kozak
Benefits of using yeast:
- easy to handle in eukaryotic system
- overproduction of proteins -> research and commercial value
- able to clone large pieces of DNA
cos ends:
12 nucleotide 5’ overhangs of linear lambda DNA cut from terminase -> long 5’ overhang means stickier than sticky ends
Ligase:
joins 5’ phosphate to 3’OH (phosphodiester bond) within double-stranded DNA using mg2+ ATP/NAD+
E.coli ligase uses
NAD+
T4 DNA ligase uses
ATP -> joins more end types
Steps of ligation
- DNA ends collide by chance (higher chance at lower temp)
- ATP/ NAD+ used to NH-AMP bond to produce PPi
- Adenyl group is transferred to 5’ phosphorylated donor
- nucleophilic attack of 3’OH on 5’ donor forms phosphodiester bond
In bactoinfection
DNA packaged inside a bacterium -> usually transient -> DNA degrades if not integrated into nuclear genome but doubling rate fast
Regulon
> 1 operon is under control of a single regulatory protein
LacZ consists of
4 identical sub-units, 2 loop at 5’end
If lactose is not hyrdolysed it’s ______
acetylated by galactoside transacetylase coded by lacA
LacZ lies next to ___ then ____
Lac Y then Lac A
Lac Y functions
lactose permease
Lac I codes for _____
lac repressor -> ihibitory -> negative control
Characteristics of lac repressor
-tetramer with a sigma 70 promoter
How does lac repressor bind DNA
binds to O2 +O3/ O1 + O3 non-specifically -> binding prevents RNAp binding and open conformation
How is the gene activated by CRP-CAP
cAMP - CRP forms in absence of glucose and binds C site near promoter -> more binding sites for CRP-cAMP which activate promoters (signalling) so RNAp binds at promoter with higher affinity
When lactose is present a small number of lactose does what to aid operon
converts to allolactose which binds tac repressor to decrease affinity
Each sub-unit of lac repressor contains an
alpha helix-turn-helix only when DNA present at ends of from hinge region
When bound repressor shape is :
1 alpha helix lies in major groove where amino acid side chains interact with bp of operator sequence
