Chemical method of transformation

Chemical method of transformation- calcium chloride mediated, Liposome mediated and PEG mediated transformation

1. METHODS OF INTRODUCTION OF
FOREIGN DNA IN HOST
Dr.Manikandan Kathirvel M.Sc., Ph.D., (NET)
Assistant Professor,
Department of Life Sciences,
Kristu Jayanti College (Autonomous),
(Reaccredited with "A" Grade by NAAC)
Affiliated to BengaluruNorthUniversity,
K. Narayanapura, Kothanur(PO)
Bengaluru 560077
CHEMICAL METHOD OF TRANSFORMATION

2. METHODS OF INTRODUCTION OF FOREIGN DNA IN HOST:
1. Chemical method: Calcium chloride mediated transformation
Polyethylene glycol (PEG) method
Liposome fusion
2. Physical method: Electroporation,
Microinjection,
Particle gun bombardment
3. Biological method: Agrobacterium mediated transformation
Viral mediated gene transfer
Screening and selection of recombinant host cells–
Direct selection and indirect selection:
-Insertional inactivation,
-in situ colony hybridization and
-Immunological assay.

3. CHEMICAL METHOD OF TRANSFORMATION:
Introduction
Cellmembraneisasheetlikeassemblyofamphipathicmolecules(havingboth
hydrophilicandhydrophobicparts)thatseparatecellsfromtheirenvironment.These
physicalstructuresallowonlythecontrolledexchangeofmaterialsamongthedifferent
partsofacellandwithitsimmediatesurroundings.
DNAisananionicpolymer,largermolecularweight,hydrophilicandsensitivetonuclease
degradationinbiologicalmatrices.Theycannoteasilycrossthephysicalbarrierof
membraneandenterthecellsunlessassisted.
VariouschargedchemicalcompoundscanbeusedtofacilitateDNAtransferdirectlyto
thecell.
Thesesyntheticcompoundsaretreatedwiththerecipientcellsandfollowedby
disturbingthecellmembranes,wideningtheporesizeandallowingthepassageofthe
DNAintothecell.
An ideal chemical used for DNA transfer should have the ability to-
• Protect DNA against nuclease degradation.
• Transport DNA to the target cells.
• Facilitate transport of DNA across the plasma membrane.
• Promote the import of DNA into the nucleus.

4. The commonly used methods of chemical transfection
use the following:
1. Calcium chloride mediated transformation
2. Polyethylene glycol (PEG) mediated transformation
3. DEAE dextran
4. Cationic Lipid-Lipofectionor Liposome mediated
transformation
5. Other polymers -poly-L-lysine (PLL),
polyphosphoester, chitosan, dendrimers

5. I.)CALCIUMCHLORIDEINDUCEDTRANSFORMATION
Introduction:CalciumchlorideinducedtransformationinvolvesmakingtheE.colicells
competenttotakeuptheplasmidDNA.SinceDNAisaveryhydrophilicmolecule,itwon't
normallypassthroughabacterialcell'smembrane.Inordertomakebacteriatakeupthe
plasmid,theymustfirstbemade"competent"totakeupDNA.
Thisinvolvesadditionofcalciumchloridetothecellsuspensionwhichenhancesthe
bindingofplasmidDNAtolipopolysaccharide(LPS).
TransformationStrainsofE.colisuchasDH5α,JM109,JM101-Competentbacteriayields5x10
6
to
2x10
7
transformedcoloniesperµgofplasmidDNA.

6. Principle:
i)Competentcellpreparation
CompetenceisaprocessbywhichacellisabletotakeinDNAfromitssurrounding
medium.Itoccursnaturallyorcanbeinducedartificiallybytreatingcellswithchemicals
suchascalciumchlorideandthenshockingthecellswithheattreatment.
Itisbelievedthatthecellwallsandmembranesofbacteriarepelthenegatively
chargedDNA.
PositivelychargedcalciumionsattractboththenegativelychargedDNAandthe
negativelychargedgroupsinthelipopolysaccharideLPSinnercoreandcanhelpattach
theDNAontothecellsurfacefollowedbyprovidingheatshockorelectricalvoltage,
cellmembranebecametransientlyporousallowingtheentryofextragenousDNA
molecules.
ii)Transformation
TheplasmidDNAcanthenpassintothecelluponheatshock,toahightemperature
(42°C)forashorttime.
ThiscausesthebacteriatotakeuptheDNA.ThecellsarethenplatedonLBagar(Luria
Bertani)mediumcontainingappropriateantibioticsafterincubationat37ºCforonehr,
toenablethetransferredgenestoexpressinE.colionrecoveryofcells.

7. CALCIUM CHLORIDE INDUCED
TRANSFORMATION

8. MATERIALS AND METHODS:
•Calcium chloride (0.1 M)
•Nutrient agar plates containing ampicillin (100 µg/ml)
•E. coliCulture DH5α
•Plasmid (pBSKS
+
)
•Nutrient Broth (NB)
TransformationStrainsofE.colisuchasDH5α,JM109,JM101-Competentbacteriayields5x10
6
to
2x10
7
transformedcoloniesperµgofplasmidDNA.

9. PROCEDURE:
Competent cell preparation and Transformation
1.A single colony of E. coli(DH5α)strain was inoculated in 5 ml of NB broth and
incubated at 37ºC, 120 rpm for overnight.
2.Then 1% of the above inoculum i.e., 1000 µl was transferred in 100 ml of LB broth.
3.It was then incubated for 2-3 hours at 37ºC till the O.D
600nm reached 0.4-0.6.
4.It was transferred into sterile falcon tube (30 ml) under sterile condition.
5.Then it was centrifuged at 3000-5000 rpm for 5-10 minutes at 4ºC.
6.Then the cell pellet was re-suspended in 20 ml of ice cold Calcium chloride (0.1 M)
and centrifuged at 3000-5000 rpm for 5-10 minutes at 4ºC.
7.Then the supernatant was discarded and the final cell pellet was suspended in 1.0 ml
of ice cold calcium chloride.
8.Then 200 µl of the above suspension was transferred into the series of eppendorf
(sterile) and stored at -20 ºC or used directly for transformation.
9.The 100 µl of competent cells were taken in fresh sterile tube and 5 µl of pBSKS
+
(~20-
100 ng) plasmid containing amp
R
gene was added and incubated on ice for 30
minutes..
10.Then it was quickly transferred to water bath maintained at 42ºC for 90 seconds (heat
shock)
11.Immediately after heat shock, 0.5 to 1.0 ml of NB broth was added to the above tube.
12.The tubes were incubated at 37ºC shaker for 60 -120 minutes.
13.100 µl of transformed cells was plated on to the NB agar containing ampicillin.

10. Calculation of Transformation efficiency
The transformation of plasmid in E. coli was assessed by counting the number of
colonies in Nutrient agar plates containing ampicillin antibiotic. The transformation
efficiency of the prepared competent cells of E. coliis calculated using:
Transformation efficiency = Number of colonies counted / µg of DNA plated
/per ml of transformed cell.
Eg. -No. of colonies obtained -500 colonies in 100 ulcells plated
-ngof DNA tranformedis 50 ng.
500 x 1000 ul= 5000
100 ul
5000 X 1000 ng= 100000 (1 x 10
5 transformants)
50 ng
ItisoneofthemostcommonmethodusedfortransformationinE.colisince
thisissimpleandcompetentcellscanbepreparedinlaboratoryandarealso
commerciallyavailablefromanumberofsuppliers.
Transformationefficienciesof10
5
to10
9
transformants/μgofDNAcaneasily
beachieved.

11. 2.POLYETHYLENEGLYCOL(PEG)-MEDIATEDTRANSFORMATION
Introduction
ThereareanumberofmethodsforintroducingDNAintoplantprotoplasts,butthemost
commonlyusedtechniqueisthepolyethyleneglycol(PEG)mediatedDNAuptake.
ThePEG-mediatedtransformationissimpleandefficient,allowingasimultaneous
processingofmanysamples,andyieldsatransformedcellpopulationwithhighsurvival
anddivisionrates.Themethodutilizesinexpensivesuppliesandequipments,andhelpsto
overcomeahurdleofhostrangelimitationsofAgrobacterium-mediatedtransformation.
ThedirectuptakeofnakedDNAbyplantprotoplastshasbeenthesolealternative
toAgrobacteriumtumefaciens-mediatedgenetransfer.
Thefirstexperimentsdemonstratingdirectgenetransferincludedthedeliveryofisolated
plasmidDNAtoprotoplastsofpetuniaandtobaccointhepresenceofpoly-L-ornithineor
polyethyleneglycol(PEG).ThePEG-mediatedDNAtransfercanbereadilyadaptedtoa
widerangeofplantspeciesandtissuesources.

13. Procedure:
ThePEG-mediatedDNAtransfermethodisamostreliablemethodforgene-targeting.
1.PlantprotoplastcanbetransformedwithnakedDNAbytreatmentwithPEGinthe
presenceofdivalentcations.e.g.,Calcium.
2. PEG and divalent cationsdestabilize the plasma membrane of the plant protoplast and
rendered it permeable to naked DNA.PEG acts as a fusogen
3.Chemicalsused,i.e.Polyethyleneglycolstimulate“Endocytosis”andthereforeDNA
uptakeoccurs.
4. The target protoplast are put in the solution containing Polyethylene glycol (PEG).
5. Exposure of protoplast to exogenous DNA in the presence of PEG allows the DNA enters
the nucleus and integrates into the host genome.
7. Intact surviving protoplasts are then cultured to form cells with cell wall.
8. The method has been successfully applied to Petunia, Nicotiana, rice, maize etc.,

14. DisadvantageandadvantagesofPEG:
1.Regenerationoffertileplantsfromprotoplastsisaproblematicforsomespecies
becauseofsecondarymetabolitesaccumulationoranycompounds.
2.TheDNAusedfortransformationisalsosusceptibletodegradationand
rearrangement.
3.Despitethelimitations,thetechniquehavetheadvantagei.e.,protoplastcanbe
isolatedandtransformedinnumberofplantsspecies.

15. III.LIPOSOMEMEDIATEDGENETRANSFORMATION
Lipofection
•Lipofectionisamethodoftransformationfirstdescribedin1965asamodelof
cellularmembranesusingliposomes.
•Liposomesareartificialphospholipidvesiclesusedforthedeliveryofavarietyof
moleculesintothecells.Theymaybemulti-lamellarorunilamellarvesicleswithasize
rangeof0.1to10micrometeror20-25nanometersrespectively.
•TheycanbepreloadedwithDNAbytwocommonmethods-membrane-membrane
fusionandendocytosisthusformingDNA-liposomecomplex.
Thiscomplexfuseswiththeprotoplaststoreleasethecontentsintothecell.
Animalcells,plantcells,bacteria,yeastprotoplastsaresusceptibletolipofection
method.
Themostfrequentlyusedphospholipidsforliposomespreparationare
phosphatidylcholines(PC),phosphatidylethanolamines(PE),phosphatidylserines(PS)
andphosphatidylglycerols(PG),DOPE,DOPS

16. Classification of Liposomes:
LiposomescanbeclassifiedaseithercationicliposomeorpH-sensitive.
Cationic liposomes-Positively charged liposomes:
•Cationicliposomesarepositivelychargedliposomeswhichassociatewiththenegatively
chargedDNAmoleculesbyelectrostaticinteractionsformingastablecomplex.
•Dioleoylphosphatidylethanolamine(DOPE)ordioleoylphosphatidylcholine(DOPC)are
somecommonlyusedco-lipids.
•ThenegativelychargedDNAmoleculeinteractswiththepositivelychargedgroupsofthe
DOPEorDOPC.
Anionic liposomes-Negatively charged liposomes:
•GenerallypH-sensitiveornegatively-chargedliposomesarenotefficientforgene
transfer.
•Theydonotformacomplexwithitduetorepulsiveelectrostaticinteractionsbetween
thephosphatebackboneofDNAandnegativelychargedgroupsofthelipids.
•However,formationoflipoplex,acomplexbetweenDNAandanioniclipidscanoccurby
usingdivalentcations(e.g.Ca2+,Mg2+,Mn2+andBa2+)

17. Lipoplex-mediatedtransfectionandendocytosis.
1.CationiclipidsformingmicellarstructurescalledliposomesarecomplexedwithDNAtocreate
lipoplexes.
2.Thestructuresfusewiththecellmembrane,afterinteractionswithsurfaceproteoglycans.
3.Thecomplexesareinternalisedbyendocytosis,resultingintheformationofadouble-layerinverted
micellarvesicle.
4.Duringthematurationoftheendosomeintoalysosome,theendosomalwallmightrupture,
releasingthecontainedDNAintothecytoplasmandpotentiallytowardsthenucleus.
5.DNAimportedintothenucleusmightresultingeneexpression.Alternatively,DNAmightbe
degradedwithinthelysosome.

18. Advantages
• Economic
• Efficient delivery of nucleic acids to cells in a culture dish.
• Delivery of the nucleic acids with minimal toxicity.
• Protection of nucleic acids from degradation.
• Measurable changes due to transfectednucleic acids in sequential processes.
• Easy to use, requirement of minimal steps and adaptable to high-throughput systems.
Disadvantages
• It is not applicable to all cell types.
• It fails for the transfectionof some cell lines with lipids.