APPENDIX F - Bacterial Transformation and Transfection
Bacterial transformation is the process by which bacterial cells take up naked DNA molecules. If the foreign DNA has an origin of replication recognized by the host cell DNA polymerases, the bacteria will replicate the foreign DNA along with their own DNA. When transformation is coupled with antibiotic selection techniques, bacteria can be induced to uptake certain DNA molecules, and those bacteria can be selected for that incorporation. Bacteria which are able to uptake DNA are called "competent" and are made so by treatment with calcium chloride in the early log phase of growth. The bacterial cell membrane is permeable to chloride ions, but is non-permeable to calcium ions. As the chloride ions enter the cell, water molecules accompany the charged particle. This influx of water causes the cells to swell and is necessary for the uptake of DNA. The exact mechanism of this uptake is unknown. It is known, however, that the calcium chloride treatment be followed by heat. When E. coli are subjected to 42degC heat, a set of genes are expressed which aid the bacteria in surviving at such temperatures. This set of genes are called the heat shock genes. The heat shock step is necessary for the uptake of DNA. At temperatures above 42degC, the bacteria's ability to uptake DNA becomes reduced, and at extreme temperatures the bacteria will die.
The process for the uptake of naked plasmid and bacteriophage DNA is the same; calcium chloride treatment of bacterial cells produces competent cells which will uptake DNA after a heat shock step. However, there is a slight, but important difference in the procedures for transformation of plasmid DNA and bacteriophage M13 DNA. In the plasmid transformation, after the heat shock step intact plasmid DNA molecules replicate in bacterial host cells. To help the bacterial cells recover from the heat shock, the cells are briefly incubated with non-selective growth media. As the cells recover, plasmid genes are expressed, including those that enable the production of daughter plasmids which will segregate with dividing bacterial cells. However, due to the low number of bacterial cells which contain the plasmid and the potential for the plasmid not to propogate itself in all daughter cells, it is necessary to select for bacterial cells which contain the plasmid. This is commonly performed with antibiotic selection. E. coli strains such as GM272 are sensitive to common antibiotics such as ampicillin. Plasmids used for the cloning and manipulation of DNA have been engineered to harbor the genes for antibiotic resistance. Thus, if the bacterial transformation is plated onto media containing ampicillin, only bacteria which possess the plasmid DNA will have the ability to metabolize ampicillin and form colonies. In this way, bacterial cells containing plasmid DNA are selected.
The transformation of bacteriophage M13 into bacterial cells is identical to plasmid DNA transformation through the heat shock step. After the heat shock step, single stranded M13 DNA begins replicating in the host cell through use of the host cell machinery. During the life cycle of this virus, however, M13 replicative form is created and daughter phages are packaged and extruded from the bacterial cell. These intact phage molecules then infect neighboring bacteria in a process called transfection. When these transformed and transfected bacteria are plated with non-infected cells onto growth media, the non-infected cells form a background cell lawn which covers the plate. In regions of M13 transfection, areas of slowed growth, called plaques, can be identified as opaque regions which interrupt the lawn.
Since M13 viral transfection is a critical part of the transformation of bacterial cells with M13, it is absolutely necessary to use a strain of E. coli which harbors the episome for the F pilus. When M13 phages infect bacterial cells they attach to the F pilus, and the loss of this pilus is a common reason for a failed or poor transformation/transfection of M13. JM101 is a strain of E. coli which possesses the F pilus if the culture is maintained under appropriate conditions. Since the F pilus is not necessary for plasmid DNA transformation, it is advisable to use GM272, a much healthier, F- strain of E. coli for this procedure. To avoid confusion between the similar procedures, bacterial transformation with plasmid DNA is termed a "Transformation", and a bacterial transformation with naked M13 followed by a transfection with intact M13 phage is called a "Transfection."
An additional level of selection can be achieved during transformation and transfections. Bacterial cells containing plasmids with the antibiotic resistance gene are selected in bacterial transformations, and cells in an area of M13 infection are recognized as plaques against a lawn of non-infected cells. However, the object of most transformations and transfections is to clone foreign DNA of interest into a known plasmid or viral vector and to isolate cells containing those recombinant molecules from each other and from those containing the non-recombinant vector. The E. coli lacZ operon has been incorporated into several cloning vectors, including plasmid pUC and bacteriophage M13. The polylinker regions of these vectors was engineered inside of the lacZ gene coding region, but in a way not to interrupt the reading frame or the functionality of the resultant lacZ gene protein product. This protein product is a galactosidase. In recombinant vectors which have an insert DNA molecule cloned into one of the restriction enzyme sites in the polylinker, this insert DNA results in an altered lacZ gene and a non-functional galactosidase. The presence or absence of this protein can easily be determined through the use of a simple chromogenic assay using IPTG and X-Gal. IPTG is the lacZ gene inducer and is necessary for the production of the galactosidase. The usual substrate for the lacZ gene protein product is galactose, which is metabolized into lactose and glucose. X-Gal is a colorless, modified galactose sugar. When this molecule is metabolized by the galactosidase, the resultant products are a bright blue color.
When IPTG and X-Gal are included in a plasmid DNA transformation, blue colonies represent bacteria harboring non-recombinant pUC vector DNA since the lacZ gene region is intact. IPTG induces production of the functional galactosidase which cleaves X-Gal and results in a blue colored metobolite. It follows that colorless colonies contain recombinant pUC DNA since a nonfunctional galactosidase is induced by IPTG which is unable to cleave the X-Gal. Similarly, for bacteriophage transfections, colorless plaques indicate regions of infection with recombinant M13 viruses, and blue plaques represent infection with non-recombinant M13.
Host Mutation Descriptions:
ara Inability to utilize arabinose.
deoR Regulatory gene that allows for constitutive synthesis for genes involved in deoxyribose synthesis. Allows for the uptake of large plasmids.
endA DNA specific endonuclease I. Mutation shown to improve yield and quality of DNA from plasmid minipreps.
F' F' episome, male E. coli host. Necessary for M13 infection.
galK Inability to utilize galactose.
galT Inability to utilize galactose.
gyrA Mutation in DNA gyrase. Confers resistance to nalidixic acid.
hfl High frequency of lysogeny. Mutation increases lambda lysogeny by inactivating specific protease.
lacI Repressor protein of lac operon. LacI[q]is a mutant lacI that overproduces the repressor protein.
lacY Lactose utilization; galactosidase permease (M protein).
lacZ b-D-galactosidase; lactose utilization. Cells with lacZ mutations produce white colonies in the presence of X-gal; wild type produce blue colonies.
lacZdM15 A specific N-terminal deletion which permits the a-complementation segment present on a phagemid or plasmid vector to make functional lacZ protein.
Dlon Deletion of the lon protease. Reduces degradation of b-galactosidase fusion proteins to enhance antibody screening of l libraries.
malA Inability to utilize maltose.
proAB Mutants require proline for growth in minimal media.
recA Gene central to general recombination and DNA repair. Mutation eliminates general recombination and renders bacteria sensitive to UV light.
rec BCD Exonuclease V. Mutation in recB or recC reduces general recombination to a hundredth of its normal level and affects DNA repair.
relA Relaxed phenotype; permits RNA synthesis in the absence of protein synthesis.
rspL 30S ribosomal sub-unit protein S12. Mutation makes cells resistant to streptomycin. Also written strA.
recJ Exonuclease involved in alternate recombination pathways of E. coli.
strA See rspL.
sbcBC Exonuclease I. Permits general recombination in recBC mutants.
supE Supressor of amber (UAG) mutations. Some phage require a mutation in this gene in order to grow.
supF Supressor of amber (UAG) mutations. Some phage require a mutation in this gene in order to grow.
thi-1 Mutants require vitamin B1(thiamine) for growth on minimal media.
traD36 mutation inactivates conjugal transfer of F' episome.
umuC Component of SOS repair pathway.
uvrC Component of UV excision pathway.
xylA Inability to utilize xylose.
Restriction and Modification Systems
dam DNA adenine methylase/ Mutation blocks methylation of Adenine residues in the recognition sequence 5'-G*ATC-3' (*=methylated)
dcm DNA cytosine methylase/Mutation blocks methylation of cytosine residues in the recognition sequences 5'-C*CAGG-3' or 5'-C*CTGG-3' (*=methylated)
hsdM E. coli methylase/ Mutation blocks sequence specific methylation A[N6]*ACNNNNNNGTGC or GC [N6]*ACNNNNNNGTT (*=methylated). DNA isloated from a HsdM[-] strain will be restricted by a HsdR[+]host.
hsd R17 Restriction negative and modification positive.
(rK[-], mK[+]) Allows cloning of DNA without cleavage by endogenous restriction endonucleases. DNA prepared from hosts with this marker can efficiently transform rK[+ ]E. coli hosts.
hsdS20 Restriction negative and modification negative.
(rB[-,] mB[-]) Allows cloning of DNA without cleavage by endogenous restriction endonucleases . DNAprepared from hosts with this marker is unmethylated by the hsdS20 modificationsystem.
mcrA E. coli restriction system/ Mutation prevents McrA restriction of methylated DNA of sequence 5'-C*CGG (*=methylated).
mcrCB E. coli restriction system/ Mutation prevents McrCB restriction of methylated DNA of sequence 5'-G[5]*C, 5'-G[5h]*C, or 5'-G[N4]*C (*=methylated).
mrr E. coli restriction system/ Mutation prevents Mrr restriction of methylated DNA of sequence 5'-G*AC or 5'-C*AG (*=methylated). Mutation also prevents McrF restriction of methylated cytosine sequences.
Other Descriptions:
cm[r] Chloramphenicol resistance
kan[r] Kanamycin resistance
Tetracycline resistance
Streptomycin resistance
Indicates a deletion of genes following it.
Tn10
A transposon that normally codes for tetrTn5
A transposon that normally codes for kan[r]
spi[-] Refers to red[-]gam[-]mutant derivatives of lambda defined by their ability to form plaques on E. coli P2 lysogens.
Reference: Bachman, B.J. (1990) Microbiology Reviews 54: 130- 197.
C600 - F-, e14, mcrA, thr-1 supE44, thi-1, leuB6, lacY1, tonA21, [[lambda]] [-]
-for plating lambda (gt10) libraries, grows well in L broth, 2x TY, plate on NZYDT+Mg.
-Huynh, Young, and Davis (1985) DNA Cloning, Vol. 1, 56-110.
DH1 - F[-], recA1, endA1, gyrA96, thi-1, hsdR17 (rk[-], mk[+], supE44, relA1, [[lambda]][-]
]-for plasmid transformation, grows well on L broth and plates.
-Hanahan (1983) J. Mol. Biol. 166, 557-580.
XL1Blue-MRF' - D(mcrA)182, D(mcrCB-hsdSMR-mrr)172,endA1, supE44, thi-1, recA, gyrA96, relA1, lac, l-, [F'proAB, lac I[q]ZDM15, Tn10 (tet[r])] -For plating or glycerol stocks, grow in LB with 20 ug/ml of tetracycline. For transfection, grow in tryptone broth containing 10 mM MgSO4 and 0.2% maltose. (No antibiotic--Mg2+ interferes with tetracycline action.) For picking plaques, grow glycerol stock in LB to an O.D. of 0.5 at 600 nm (2.5 hours?). When at 0.5, add MgSO4 to a final concentration of 10 mM.
SURE Cells - Stratagene - e14(mcrA), D(mcrCB- hsdSMR-mrr)171, sbcC, recB, recJ, umuC::Tn5 (kan[r]), uvrC, supE44, lac, gyrA96, relA1, thi-1, end A1[F'proAB, lacI[q]DM15, Tn10(tet[r])]. An uncharacterized mutation enhances the a[-] complementation to give a more intense blue color on plates containing X-gal and IPTG.
GM272 - F[-], hsdR544 (rk[-], mk[-]), supE44, supF58, lacY1 or [[Delta]]lacIZY6, galK2, galT22, metB1m, trpR55, [[lambda]][-]
-for plasmid transformation, grows well in 2x TY, TYE, L broth and plates.
-Hanahan (1983) J. Mol. Biol. 166, 557-580.
HB101 - F[-], hsdS20 (rb[-], mb[-]), supE44, ara14, galK2, lacY1, proA2, rpsL20 (str[R]), xyl-5, mtl-1, [[lambda]][-], recA13, mcrA(+), mcrB(-)
-for plasmid transformation, grows well in 2x TY, TYE, L broth and plates.
-Raleigh and Wilson (1986) Proc. Natl. Acad. Sci. USA 83, 9070-9074.
JM101 - supE, thi, [[Delta]](lac-proAB), [F', traD36, proAB, lacIqZ[[Delta]]M15], restriction: (rk[+], mk[+]), mcrA+
-for M13 transformation, grow on minimal medium to maintain F episome, grows well in 2x TY, plate on TY or lambda agar.
-Yanisch-Perron et al. (1985) Gene 33, 103-119.
XL-1 blue recA1, endA1, gyrA96, thi, hsdR17 (rk[+], mk[+]), supE44, relA1, [[lambda]][-], lac, [F', proAB, lacIqZ[[Delta]]M15, Tn10 (tet[R])]
-for M13 and plasmid transformation, grow in 2x TY + 10 ug/ml Tet, plate on TY agar + 10 ug/ml Tet (Tet maintains F episome).
-Bullock, et al. (1987) BioTechniques 5, 376-379.
GM2929 - from B. Bachman, Yale E.coli Genetic Stock Center (CSGC#7080); M.Marinus strain; sex F[-];(ara-14, leuB6, fhuA13, lacY1, tsx-78, supE44, [glnV44], galK2, galT22, l[-], mcrA, dcm-6, hisG4,[Oc], rfbD1, rpsL136, dam-13::Tn9, xyl-5, mtl-1, recF143, thi-1, mcrB, hsdR2.)
MC1000 - (araD139, D[ara-leu]7679, galU, galK, D[lac]174, rpsL, thi-1). obtained from the McCarthy lab at the University of Oklahoma.
ED8767 (F-,e14-[mcrA],supE44,supF58,hsdS3[rB[-]mB[-]], recA56, galK2, galT22,metB1, lac-3 or lac3Y1 , obtained from Nora Heisterkamp and used as the host for abl and bcr cosmids.
Notes on Restriction/Modification Bacterial Strains:
1. EcoK (alternate=EcoB)-hsdRMS genes=attack DNA not protected by adenine methylation. (ED8767 is EcoK methylation minus). (1)
2. mcA (modified cytosine restriction), mcrBC, and mrr=methylation requiring systems that attack DNA only when it IS methylated (Ed8767 is mrr+, so methylated adenines will be restricted. Clone can carry methylation activity.) (1)
3. In general, it is best to use a strain lacking Mcr and Mrr systems when cloning genomic DNA from an organism with methylcytosine such as mammals, higher plants , and many prokaryotes. (2)
4. The use of D(mrr-hsd-mcrB) hosts=general methylation tolerance and suitability for clones with N6 methyladenine as well as 5mC (as with bacterial DNAs). (3)
5. XL1-Blue MRF'=D(mcrA)182, D(mcrCB-hsdSMR-mrr)172,endA1, supE44, thi-1, recA, gyrA96, relA1, lac, l-, [F' proAB, lacI[q]ZDM15, Tn10(tet[r]
REFERENCES:
1. Bickle, T. (1982) in Nucleases eds Linn, S.M. and Roberts, R.G. (CSH, NY) p. 95-100.
2. Erlich, M. and Wang, R.Y. (1981) Science 212, 1350-1357.
3. Woodcock, D.M. et al, (1989) Nucleic Acids Res., 17,3469-3478.