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Molecular cloning
reproducing many exact copies of a gene of interest
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The purpose of cloning
- isolation and propagation of pure DNA fragments
- prep of specific probes of DNA and/or RNA
- sequencing regions of genomes
- expression and purifications of large amounts of protein
- modification of DNA sequences to study the function of a gene
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What is a clone?
- collection of nolecules or cells, all identical to an original molecule or cell
- can be exact copies or altered versions
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Recombinate DNA cloning procedure
- cut DNA and plasmid with restriction enzymes
- ligate foreign DNA into vector with T4 DNA lygase
- introduce into the host cell
- spread on agar plate with antibiotic at 37 degrees
- pick colonies and screen colonies for insert
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DNA can be cloned using...
- chromosomal DNA
- RNA converted to cDNA
- recombinant DNA-subcloning
- PCR-amplified DNA
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What is recombinate DNA
- production of a unique DNA molecule by joning together 2 or more DNA fragments not normally associated with each other
- fragments are usually derived from different biological sources or by dynthesis and amplification using PCR
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Steps in recombinant DNA
- DNA molecules digested
- digestion inserted into vector
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Vectors.....
- carriers of the forging DNA (usually plasmids)
- have origin of replication, unique restiction enzymes sites, and selectable markers
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Plasmids
- extrachromosomal DNA (1000 - 10,000 bp)
- circular dsDNA
- can be cleaved by restriction enzymes leaving sticky or blunt ends
- artificials can be constructed by linking new DNA fragment to sticky/blunt ends
- replicated independently of bacterial chromosomes and can move into bacterial cells
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Good cloning vectors are....
- small and easy to manipulate
- easy to transfer from cell to cell
- easy to isolate for the host organism
- easy to detect and select
- multiple copies help obtain large amounts of DNA
- polylinkers allow insertion of cloned DNA
- method to detect presence of inserted DNA
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Bam HI and Bgl II gernerate the _____ overhanging or sticky ends
- same; 3'-CTAG-5' and 5'-GATC-3'
- the 2 ends are complementry and base pair
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Treating a plasmid twih alkaline phosphatase will prevent religation by
removing the 5' phosphate and prevent the plasmid from recircularizing, also reduces background colonies caused by recircularizing.
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Non directional cloning
- plasmid and gene of interest cut with same restriction enzyme
- gene can go into the plasmid in either direction
- can do digest and colonie PCR to determine if the plasmid is in the correct direction
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Directional coloning
- make 2 cleavages with 2 different restriction enzymes
- construct foreign DNA with same restriction enzymes
- foreign DNA can only be inserted in one direction
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Transformation
uptake and stable incorporation of DNA element, usually is introduction of plasmid into bacteria
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Compentent cells
chemically treated E. coli to allow feeicent uptake of DNA (CaCl)
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Lac Operon
consits of 3 structural genes lacZYA that are transcribed from the lacP promoter
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lacZYA
transcribed as a polycistronic message
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lac operon repressor
upstream of the promoter in the LacI protein
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LacZ gene encodes ______ which cleaves lactose into ______ and ______
beta-galactosidase; galactose;glucose
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LacY gene encodes for _____ with transports _____ across the cytoplasmic membrane into the bacteria
lactose permerase; lactose
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LacA gene encodes for _____ with an unknown role
Lactose acetylase
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The Lac promoter has a binding site for _____ which overlaps the binding site for RNA polymerase. This region is also called the _____ and prevents transcritpion
lacO; operator
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The global regulator that activates transcription of many different operons for using alternate sugar sources. It is active when E. Coli does not have glucose to utilizw as an energy source
CRP protein
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When E. coli has plenty of glucose the lactose operon is ___
off
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When glucose is present
cAMP is low
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When E. coli runs out of glucose cAMP levels ______ and bind _____
increase; CRP
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If lactose is available beta-gal catalyzes a side reaction, converting some lactose into ______ and acts as an ______ and bind to the lacI repressor protein
allolactose; inducer
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Insted of allolactose, _____ is used in the lab because it is not cleaved by beta-gal because it is linked through sulfur insead of oxygen
IPTG
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IPTG is a nonmetabolizable _____ of allolactose
analog
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Lactose operon is turned on whe glucose is ____ but lactos is ____
absent; present
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When glucose is available, cAMP is ____ and CRP _______ binding of RNA polymerase
low; does not activate
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When there is ______ glucose, cAMP is high and binds to ____
no;CRP
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CRP-cAMP binds to the promoter and ______ RNA polymerase to bind
stimulates
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When there is no lactose, the LacI protein is bound to the operator sie and prevents______
transcription
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Allolactose is transcribed and lacI-allolactose complex is released in the presence of ______
Lactose
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What is the Alpha complementatin selection method
takes advantage of the lac promoter to determine which colonies cntain plasmids with inserts
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In the present of alpha complementation
lacZ-alpha and lacZ-omega combine to produce acitve enzymes and colonies turn blue
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In the absence of alpha complementation
the lacZ-alpha is interrupted and omega is inactive and colonies remain white
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pBR322 bacterial vectors
- first plasmid created in the lab
- small and easily isolated
- accommodate DNA up to 5-10 kb
- several unique restriction sites
- encodes ampicillin and tetracyclin resistance genes
- if resistance genene is broken bacteria receiving the plasmid will be sensitive to the antibiotic and die if treated with antibiotic
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Important elements in expression vector
- Promoter: -35(TTGACA) and -10 (TATAAT) region
- terminator (hairpin)
- rigosome binding site (RBS)
- selection marker
- vextor replication seq (ori)
- polylinker (MCS)
- start (ATG)
- stop (TAA, TAG, TGA)
- signal peptide
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______ are hybrid molecules designed for use in multiple cell types
shuttle vectors
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_______ allow replication in both prokaryotic and eukaryotic host cell allowing transfer between different cell types
Multiple ORIs
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This vector can survive in either bacteria or yeast because it has both yeast and bacterial origins of replication
yeast shuttle vector
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Because ________ is easy to grow and manipulate, its gemone has been modified to accept foreing DNA inserts
lambda phage
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______ can hold pieces of DNA up to 45 kb in length
cosmid vectors
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Cosmid vectors are ______ with all the sequences between the cos sites removed and replaced with the insert
highly modified lambda vectors
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