Cloning and expression vectors lecture 9 and 10 Flashcards
What are vectors used for in molecular biology
They are used to generate a wide range of recombinant proteins.
Cloning vectors
A cloning vector allows you to ‘store’ your GOI and replicate it in E.coli.
Types of cloning vectors
-plasmid vector (pBR322, pUC18/19)
- Cosmic vector (pJB8)
-Phagemid vector (pEMBL8)
-Bacteriophage vector (M13)
-Artificial chromosome (PAC, BAC, YAC)
Expression vector `
An expression vector additionally facilitates transcription and translation of the GOI, thus leading to the production of the encoded ‘recombinant protein’, which can then be purified.
There are expression vectors tailored for use in E.coli, yeast, or mammalian cells.
why does the MCS have many unique RS
to allows you to insert your DNA fragmen
what is the function if the Ampicillin resistance gene
Ampicillin resistance gene allows you to isolate bacteria that have taken up the plasmid
what is the use of the Origin of replication
This allows the plasmid to replicate independently to high copy numbers in E.coli when it is taken up, replicating also the insert we put into it.
what is the importance of the Ampicillin resistance gene
It is used to isolate E.coli that have taken up the plasmid as plasmid transformation is inefficient
what is the process involved in isolating ecoli cells that have taken up the plasmid using ampicillin
We spread transformed cells on LB Agar plates with an appropriate antibiotic after transformation (in this case Ampicillin, but could be Kanamycin or Chloramphenicol).
what is blue white screening used for
Bue-white screening allows us to identify colonies with an insert in the MCS
what is the function of X-Gal and IPTG in Blue-white screening
X-Gal is a chromogenic substrate for beta-galactosidase that yields a blue precipitate upon hydrolysis, while IPTG induces the transcription of genes from the lac operon in bacteria, notably the hydrolase enzyme beta-galactosidase.
why is cdna used in PCR involving Ecoli.
Ecoli cannot handle intron containing genes as intros present in genomic DNA are too big
How is cDNA produced and used.
mRNA is isolated from cells and then reverse transcribe into cDNA. This cDNA serves as a template in PCR and then the amplified PCR product is inserted into an appropriate expression vector.
what do Expression vectors allow
they allow cloned genes to be transcribed and translated in a
host cell.
what are the two types of expression vectors
bacterial expression vectors and mammalian expression vectors
In expression vectors what is incorporated upstream of the insert gene product and what does It facilitate.
expression vectors have to incorporate a promoter and elements upstream of the inserted gene product to facilitate transcription and ranslation initiation of the resulting mRNA, respectively.
in Transcription and Translation in an expression vector what is the purpose of the promoter region
- should facilitate high level constitutive transcription of the GOI
- Can use promoters of highly expressed house-keeping genes
(e.g. Ubiquitin for mammalian expression vectors) - Often viral promoters are used
- e.g. bacteriophage T7 promoter for expression in E.coli - or CMV for expression in mammalian cells
in Transcription and Translation in an expression vector what is the purpose of translation start site
- these elements mediate ribosome recruitment and/or start codon recognition when present in the transcribed mRNA
- E.g. Shine-Dalgarno Sequence for correct translation in
E.coli - E.g. Kozak Sequence for translation initiation in
mammalian cells
what are the 3 tags
-Affinity tags (for affinity purification of the protein) -Epitope tags (for recognition by epitope-tag antibodies, facilitating detection and immunoprecipitation)
-Fluorescent protein tags (detection, localisation)
what are the affinity tags for protein purification
GST-tag: Glutathion-S-transferase enzyme, which was purified using Glutathion- Sepharose, the substrate for the enzyme.
His-tag: a stretch of 6 Histidine residues that can be purified by binding to Nickel-Agarose
characteristics of Epitope tags for easy detection of your protein
Epitope tags are generally shorter in length than affinity tags and are, therefore, less likely to affect protein function.
* Epitope-tagged proteins can easily be detected with antibodies raised to these epitopes.
* They can also be ‘immunoprecipitated’ (for separation from other proteins) using these antibodies coupled to a solid matrix.
* Although they can be used for affinity purification, the columns are based upon immobilized antibodies, which are usually more costly or not as efficient as columns for affinity tags.
where and was the Green Fluorescent Protein first isolated
from Jellyfish Aequorea victoria in 1962.
where and how can tagged proteins be expressed
Tags can be added to the C-terminal or N-terminal end of your gene of interest (depending on position in the vector).
For C-terminal tags, make sure to remove STOP codon in the sequence of your gene of interest and maintain correct reading frame!!
For N-terminal tags, make sure you maintain correct reading frame when inserting your gene of interest!
Inducible expression vectors
allow us to control the expression of our target gene in E.coli (i.e. we can switch it on when we want).
use of lactose by Ecoli
Lactose is the main sugar in milk. Lactose can be an excellent energy source for E. coli bacteria. However, they’ll only use lactose when other, better sugars –
like glucose – are unavailable.
Lac operon
The lac operon is an operon, or group of genes with a single promoter (transcribed as a single mRNA).
* The genes in the operon encode proteins that allow the bacteria to use lactose as an energy source.
Two regulators turn the operon “on” and “off” in response to lactose and glucose levels
- the lac repressor protein acts as a lactose sensor and normally blocks transcription of the operon but stops acting as a repressor when lactose is present
- catabolite activator protein (CAP) senses lactose indirectly, through its isomer allolactose.
what 3 genes are in the lac operon
lacZ, lacY, and lacA.
what is the purpose of the Genes in the lac operon
These genes are transcribed as a single mRNA, under control of one promoter.
Genes in the lac operon specify proteins that help the cell utilize lactose.
lacZ
lacZ encodes an enzyme (beta-galactosidase) that splits lactose into monosaccharides (single-unit sugars) that can be fed into glycolysis.
lacY
encodes a membrane-embedded transporter that helps bring lactose into the cell.
lacA
lacA encodes an enzyme that helps clean up any toxic metabolic by-products of lactose metabolism
regulatory DNA sequences in the lac operon
These are regions of DNA to which particular regulatory proteins can bind, controlling transcription of the operon.
Inducible expression vectors contain lac Operon elements
In the absence of lactose a repressor protein binds to the promote suppressing the lac promotor
If present, lactose binds to repressor protein, which then detaches from the promoter, so that transcription can now occur (gene expression induced by lactose).
In the expression vector, our inserted gene is placed under the control of the lac promoter/operon and its expression can therefore be switched on by adding IPTG (a lactose analogue).
what does the T7 promoter under the control of the lac operator ensures
T7 promoter under the control of the lac operator ensures that expression of our inserted gene product can be regulated.
Normally repressed by lac repressor protein, but can be de-repressed by adding IPTG
what is the T7 promoter transcribed by
T7 promoter is only transcribed by T7 RNA polymerase, a gene encoded by the T7 phage. High transcription rate. Means the T7 RNA polymerase also needs to be introduced into E.coli as it does not naturally occur in these cells.
