Week 5: Molecular Cloning Strategies

Transcription

1 Week 5: Mlecular Clning Strategies - 2 ptins fr gene clning: Traditinal recmbinant plasmid clning f the gene f interest PCR amplificatin f the gene f interest within bkended primers - Bth yield many cpies f the gene same utcme - In bth, the gene cassette may be mved arund Recmbinant Plasmid - A vectr is linearised by digesting it at a unique restrictin site, whilst preparing a surce f linear insert DNA with the same r cmpatible restrictin ends E.g. vectr + insert prepared with enzyme EcRI - Other ways f ding this: Using enzymes that are isschizmers f ne anther Using any blunt-ended restrictin enzyme Using tw different enzymes - First 2 chices the inserts will be aligned in either directin relative t the vectr - Last 2 chices the new site in the recmbinant plasmid might nt be able t be cut with either riginal enzyme Psitive Selectin - Only 2 types f mlecules are imprtant + they are thse that can be transfrmed: Recircularised vectrs (blue circles) Recmbinant circles (grey)

2 - The backgrund f vectr and varius recmbinants is unimprtant bc we have a selectable phentype P fr the clne we want Phentype is easily identified e.g. antibitic resistance r clny clur E.g. if phentype is kanamycin resistance, nly need t use agar plates with ampicillin and kanamycin t btain the required recmbinant clnes Nt as easy in practice, may need t g thrugh thusands f transfrmants t find the recmbinant clne f interest - Want t reduce r eliminate the backgrund s desired clnes will be present - Successful mlecular clning = generating ONLY recmbinant plasmids - The linear cncatamers are rendered redundant by using ideal ligatin cnditins Prblem = backgrund f riginal vectr plasmids that will represent the majrity f transfrmants (~60-90%) depending n the sizes f the inserts in the recmbinant plasmids Bc small plasmids transfrm much mre efficiently than larger nes Rate abut 1 lg less per dubling in plasmid size Insertinal Inactivatin - II f antibitic resistance gene = discriminate bet religated vectrs and recmbinants - E.g. a DNA linear fragment with BamHI ends is ligated int the unique BamHI site in the tetracycline resistance gene. 2 results: Recmbinant plasmid result = susceptible t tetracycline bc the tet gene ORF sequence has been interrupted and the Tet prtein cannt be made cannt elicit tetracycline resistance Original vectrs that religate withut an insert = still express Tet resistance

3 Velvet Press - Grid plating methd t screen clnes frm insertinal inactivatin - A master grid f transfrmants is patched nt an Amp-nly agar plate Plate is velvet-pressed nt a Tet-nly agar plate - Only vectr transfrmants ( backgrund ) grw n the Tet plate Actual recmbinants are thse that did nt grw These may be recvered frm the master plate (red clnes) Determine by psitin n plate (lk at last 2 plates in diagram) - In practice, methd is effective, but: Requires a special vectr (with 2 antibitic markers) Labur intensive + time cnsuming needs fairly extensive screening t btain a sizable number f recmbinants (60-90% will be vectrs) Prtein Cmplementatin 2 separate segments f the same riginal single plypeptide can assciate in the same cnfrmatin and carry ut the same functin as the riginal LacZa B-Gal Cmplementatin - Mst current vectrs (plasmids + viruses) use this beta-galactsidase selectin system fr discriminating between religated vectrs and recmbinants - Cmplementatin B-galactsidase breaks apart glycsidic bnds = blue clnies - Cmplete enzyme nly wrks as a tetramer r dimer f dimers Alpha-peptide deletin = enzyme can nly assemble as a dimer = inactive Strain f E. cli with minr deletin (30aa s) affects flding cannt assciate int tetramer, nly dimers cannt hydrlyse substrate = white clnies - Alpha-peptide supplied by vectr = cmbine with defective enzyme prduced frm hst chrmsme = generate an active tetramer by nn-cvalent cmplementatin

4 Plasmid Vectr Optimised fr Clning - Inactivated lacz regin by clning int the MCS in the recmbinant plasmid (right) Basis f selectin fr recmbinant clnes frm mst mdern vectrs - E.g. f insertinal inactivatin Mre suited t smaller vectrs Is direct (blue-white clny identificatin) vs. patched grids and extra antibitic cntaining plates Plasmid Vectr Features - Elements f the blue-white LacZ selectin system depicted in a typical clning vectr - Inserts are ligated int the MCS t generate recmbinant plasmids MCS is within the shrt lacz alpha-peptide sequence (cdns fr aa s 11-41) f the lacz gene Majr prtin being in the E. cli hst chrmsme - Vectr cntains the prmter fr lacz alpha-peptide sequence can be transcribed Must be triggered by the inducer IPTG present in agar selectin plates IPTG als induces transcriptin f the defective lacz gene fr betagalactsidase in the hst chrmsme Selectin plates als cntain: Ampicillin fr plasmid-transfrmants selectin Beta-galactsidase chrmgenic substrate X-gal prduces free indle when hydrlysed by a B-galactsidase enzyme = blue clnies - Insertinal inactivatin als wrks - Elements f the clning vectr and the hst E. cli chrmsme cmbine t generate the active enzyme by cmplementatin (Left) Religated vectrs supply an uninterrupted peptide that cmplements the defective enzyme = functinal galactsidase = hydrlyse synthetic substrate X-gal = liberating indle = blue clnies cntaining vectr plasmids

5 (Right) Insert ligated int the MCS f the vectr alpha-peptide is insertinally inactivated = defective galactsidase enzyme = white clnies cntaining recmbinants - Determined in the Amp-X-gal-IPTG agar plates the transfrmants are plated - IPTG = inducer mlecule Remves the repressr frm the peratr-prmter n the vectr puc19 Enables read thrugh uninterrupted transcriptin f the LacZ alpha-peptide Cmplements defective beta-galactsidase frm the chrmsme Enzyme is able t hydrlyse the synthetic substrate X-gal, releasing indle that gives the clnies a blue clur (vectr backgrund) - In recmbinants n hydrlysis f X-gal and n indle r blue clur = white - The blue/white system is ften teamed with ne f these vectr reductin schemes Reasn achieving cmplete vectr eliminatin is never certain There culd be 1-2% f residual vectrs that slip thrugh the system ensure that plated transfrmants are mstly recmbinants by using the blue/ white selectin agar plate system mst clnes will be white and there culd be a few blue clnies, and these are avided Central Ideas f Mlecular Clning - Maximising the efficiency f the ligatin reactin bet vectr plasmid + DNA insert - Reducing the religatin f the vectr + high vectr backgrund f transfrmants - Mdifying the restrictin ends f the vectr +/ DNA insert t enable brader and mre flexible recmbinant DNA pssibilities - Mlecular clning fr multiple cpies f genes PCR will d the same thing Mdifying DNA fr clning r research Using a vectr fr expressing prteins Zer Backgrund - Ways f cntrlling vectr backgrund: Preventing the vectr being regenerated Preventing vectr s survival in transfrmants

6 E. cli Death Gene ccdb Prduct f gene = ccdb prtein inhibits DNA gyrase = cell cannt repair r replicate its chrmsme - Vectr pzero has all f the usual regulatry elements f a clning vectr - If the lacz-ccdb MCS has a DNA fragment inserted, the ccdb sequence is interrupted by insertinal inactivatin Recmbinant plasmids will nw survive in transfrmed cells Religated vectr will result in the death f the transfrmed cell Vectr backgrund is absent and all clnies cntain recmbinant plasmids - If ccdb is a lethal gene in the vectr pzero, hw is it pssible t intrduce the plasmid int E. cli and make multiple cpies f it fr clning purpses? It will nly cause death if it is transcribed and prtein is made must als induce prmter Withut inducer, prmter is drmant = n prtein = cell survives Ligatin: Orientatin f Ends - Fr expressin studies, it is essential that the insert (the rientatin f the gene) is in the crrect directin dwnstream frm the vectr s regulatry regin and prmter Directinal/Frced Clning - Tw imprtant advantages: Reducing vectr backgrund t zer s that mst r all transfrmants will cntain recmbinant plasmids Achieved by cutting the vectr with tw different + incmpatible restrictin enzymes Insert is prepared with the same 2 incmpatible sites n either end Ligatin reactin will generate the recmbinant plasmid N backgrund bc the vectr is incapable f religating its ends cntain incmpatible restrictin sites all transfrmants will carry recmbinant plasmids Insert is ligated int the vectr in ONE (frced) directin nly all recmbinants (100%) are the same (100%) Ensures that a clned gene will have the crrect rientatin (5 t 3 ) with respect t the upstream vectr prmter

7 Alkaline Phsphatase - Methd fr reducing vectr backgrund - Alkaline phsphatase remves 5 phsphates frm the ends f DNA mlecules - E.g. vectr and insert are bth prepared with EcRI ends Linearised vectr is then treated with alkaline phsphatase = remves the 2 diagnally ppsite 5 phsphates - Vectr cannt be religated t itself = remains linear bc incapable f refrming the riginal phsphdiester bnds at the EcRI cut site vectr backgrund is reduced t zer - Phsphate-free linearised vectrs can be ligated t the insert bc the insert still has its tw 5 phsphates Circular recmbinants may be frmed with diagnally ppsite phsphdiester bnds (ne n each strand) and diagnally ppsite nnligated hydrxyl ends Half-clsed circular plasmids may be transfrmed int cmpetent E. cli cells as efficiently as fully clsed circular plasmids Newly replicated recmbinant plasmids in the cells will be fully clsed circles - Linkers and adaptrs can increase the # f clning ptins

8 Mdifying Restrictin Sites: Linkers - Cnverting restrictin enzyme blunt ends t staggered ends - Blunt-ended linkers are ligated t the ends f the fragment There is a range f linkers available that have internal restrictin sites fr mst f the cmmn enzymes that generate staggered ends - E.g. an excess f linkers is used s that all f the available ends in the fragment sample will be linked Excess f linkers drives the reactin and als results in multiple end t end (tandem) linkers being attached Hwever, when the ligated linkers are subsequently cut with EcRI, all f the added linkers are digested away Only the last tw that remain attached t the fragment remain Have designer EcRI ends Mdifying Restrictin Sites: Adaptrs - Cnverting restrictin enzyme blunt ends t staggered ends - Difference = adaptrs have ready-made restrictin enzyme staggered sites that, nce ligated t the blunt-ended fragment, require n further treatment ther than nw being used in a clning scheme adaptrs cut ut the need fr the restrictin reactin step t generate the enzyme site - Adaptrs must be dephsphrylated 5 phsphates are remved by alkaline phsphatase digestin Required t prevent the staggered ends f the adaptrs frm self-ligating, which wuld the require an additinal restrictin enzyme step End-Filling - Cnverting restrictin enzyme staggered ends t blunt ends - T4 DNA plymerase end-fills either 3 r 5 recessed staggered ends

9 Multifunctinal enzyme Adds nucletides t a single-stranded recessed 3 end (tp) r digest excess 3 nucletides (bttm) that verhang a staggered site Only adds in 5 t 3 directin if it cannt fill in, snips ff verhanging nucletides - Bth types f staggered sites can be generated by different restrictin enzymes Methylatin f Sites - Mimics the methylase-restrictin hst bacterium system - Methylase adds methyl grups t ne f the nucletide bases within the restrictin site f it s partner enzyme prevents the hst s restrictin enzyme frm digesting its wn DNA - E.g. EcRI methylase adds methyl grups t the ppsite first adenine bases acrss the EcRI site Site is n lnger a substrate fr EcRI as it is prtected frm enzyme binding and digestin In vitr step used in MC scheme that requires a DNA mlecule t be at a separate EcRI site, but nt at the nw prtected site, which culd, fr example, be within a gene sequence, whereas the nn-prtected EcRI site wuld be external t the gene