L7- Gene cloning and expression in Bacteria

what is gene cloning

making multiple copies of a gene and manipulating it

the 3 types of theraputic drug designs

how did we used to get protein of interest and problems

take cells that have genes, make transcrips and make proteins then could purify proteins made form those cells e.g. done with insulin

problems

• need somewhere to get starting material

• make sure there is a lot of it

• contamination

• ethical issues

,

what do we do now to get protein of intrest

• use moelcular genetics to isolate gene of intrest

• engeneer the gene and put it in a host cell that will happilie grow into huge quantite

• and make large amounts of that protien

• then purify that protein

what do we need to consider when picking host organism

• non pathogenic

• can easily be manipulates - allows itsself to be manipulated

• we understand the mechanism the cell uses to be able to manipulate it

• easily cultures

Recombinant DNA

• use same RE on 2 different organsism

• RE cuts at spcifci sequnces so leaves compatibe stickly ends

• and T4 DNA ligase get DNA joing from different organisms togetehr

  • have hybrid DNA

How do we amplify the recombinant DNA

• bacteria divides and divies again making multiple coppies of itself

• if put DNA of intrest into that single bacterial cell will then copy over and over again making multiple cells cinatiang recombiant DNA

where is DNA added in teh bacterium

2 places can put the dna

• bacteria has 1 chromosome, can put DNA in the chromosome but every time divides only get 1 more copy

• preferred way is to add it to plasmid - circular DNA , bacteria has multiple so when divide have much ,more copies, they are advantagous (antibiotic resistance , nitrogen fixation and virulence)

Bacteria and plasmid uptake

• as have antibiotic resiance can be tarsfered between bacteria through horizontal gene transfer

• Transformation → randomly take up plasmids in environemnt, done at really low rates, its a stress response

(other ways is by conjugation and transduction)

how do scientist increase bacteria uptake of plasmids

• using heat shock create a stressuful envoronmet for bacteria and so higher rate will pick up plasmid from environemnt

• plasmids contain antibiotic resistnace can add antibiotic to check if bacteria has taken up plasmid

• should survive when exposed to antibiotic

plasmid can also rejoin back on itself instead of joing DNA how do we stop this

remove 3’ pi group using alkali phosphatase

how do we engerneer plasmis

we add antibiotic resistanec gene and add origin of replication site → know both of tehse sequnces so can add to plamid

on a seperate are on plasmid we add the RE sites so when cuts only cuts there and not other things we add - multiple cloning site

then can add DNA

also make sure to add alkali phosphitase

How do we select the recombinat plasmids

• The plasmid contains the LacZ gene with a multiple cloning site (MCS) inside it

• Bacteria are grown on agar containing antibiotic and X-gal

• If the plasmid enters the bacteria successfully, the bacteria survive the antibiotic

• If LacZ is functional, β-galactosidase is produced

• β-galactosidase breaks down X-gal, producing a blue colour

• If foreign DNA is inserted into the MCS, the LacZ gene is disrupted

• Disrupted LacZ means no functional β-galactosidase is made

• Colonies with inserted DNA remain white

• Blue colonies = no DNA insert

• White colonies = plasmid contains inserted DNA (desired colonies)

what to do when fragmnet does not have any of the RE in it

• can use blunt ends → inefficent

• can use compatible cohesive ends

  • use PCR to add RE sites

How do we use PCR to add RE sites

• Restriction enzyme (RE) sites can be added to PCR products by designing them into the 5′ end of PCR primers

• The primer contains:

  • extra clamp bases

  • the RE recognition site

  • a region complementary to the target DNA

• Only the 3′ end of the primer needs to bind the template DNA for DNA polymerase extension to occur

• The extra 5′ sequence containing the RE site does not need to base-pair initially

• During PCR, DNA polymerase extends from the primer’s free 3′ OH end

• The extra RE site sequence becomes incorporated into the newly synthesised DNA strand

• After multiple PCR cycles, all amplified DNA products contain the RE sites at their ends

• Extra clamp nucleotides are added before the RE site because restriction enzymes cut poorly at the very end of DNA

• The PCR product can then be digested with the restriction enzyme to generate sticky ends for cloning into a plasmid cut with the same enzyme

TA cloning

• can create recombnate plasmid withou RE site

• Taq polymerase in pcr very ineffcient uslaly adds A to the 3’ ends to PCR porducts

• can get vector with T ends and then they van join togetehr as complementary

• not efficent as only 1 base pair

TOPO cloning

• much fatser than original ways when no RE sites

• palmids naturally have TOPO vector with tropoisomerae 1 attached to ends with 3’ T on it

• in PCR Taq polymerasee leaves 3’ A

• Toposimonerase joings the A and the T toegteher insead od DNA ligase

• much quicker

example of inducible system

probelm with genes in euk

• our DNA has introns, taht we splice to make mRNA

• bacteria cannot romove introns so giveing out DNA directly will not be usefl - make non fuctional proteins

  • so what we do get matur mRNA and convert it into cDNA usuing reverse tarscription

Lacz induciable system

• have a repressor that binds to lac operon so RNA polymerase cannot trnacribe

• when add allolactose binds to repressoe allowing gene to be transcribed

Protein production

• when repressor is on lac operon cannot make T7 RNA polymerase which tarnscribes gene of interest

• when add IPTG induces tarscription of T7 gene and then go on to trascribe gene of intrest

  • use gel elecropheresis to see which proteins are being and in large amounts before and after IPTG addition

Protein production and purification

• The gene of interest is cloned into an expression plasmid/vector

• A DNA sequence encoding a protein tag is added next to the gene

• Common tags include:

  • His-tag → binds nickel

  • MBP → binds maltose

  • GST → binds glutathione

• The plasmid is transformed into bacteria (e.g. E. coli)

• Bacteria are grown in culture and protein expression is induced (often using IPTG)

• The bacteria produce the tagged protein of interest

• Cells are lysed to release proteins, DNA and other cell contents - break open cells

• The lysate is loaded onto an affinity chromatography column containing beads that bind the tag

• The tagged protein binds to the beads while most other proteins flow through

• The column is washed to remove unbound contaminants

• Elution buffer releases the tagged protein from the beads

• Purified protein fractions are collected

Bacteriaphage