Note: Descriptions are shown in the official language in which they were submitted.
2 - HOE 8~/F 1~7 K
The invention relates to the ~ene~ic manipula- .
tion o~ me~hylotrophic microorganisms~ in par~icular pro~
cesses ror the preparcition of obligate methyLotrophic
bac~eria which express contained foreign DNA, plasmids
for introducing the ~oreign DNA, and hos~ or,~Janisms. One
of ~he proce7,ses according ~o the inven~ion comprises
a~ ;sola~in~ a plasrnid which orisinates from cin obligate
me~hylotrophic ~arterium,
b) preparing from ~his and ~rom a plasmid wi~h selection
10 markers a hybrid plasmid w;th a repl;con inheren~ ~o ~he
obligate methylo~rophic ~acterium,
c) introducing ~hls hybrid plasmid by transform2tion in~
~o a hos~ organism and ampl;fy;ng i~ ~here,
d) a~ter selec~;on, trea~;ng the clones w;~h a su;table
~5 conjugat;ve plasm-id ancl abol;sh;ng the mob;li~ability
defect of ~he hybr;o plasmld,
e) conjuga~;ng the clones ~hus obtained wi~h obligate
methylotrophic bac~er;a as the rec;p;erit and
f) selec~ing ~he des;re~ clones
r
lhe plasmid ernployeci ;n process step a) is pre~
~er~abiy isola~ed ~rom a ~ac~erium of ~he~~enus '.~'e~hy~
omonas, in particular o~ ~he spec;es ~e~hylomonas clara.
The plasmic, p~E 3 from ~he M~ra~hylorilonas clara strain
which is deposiLed in .~,e Germa,i Collec~ion of Microorgan- !
Z5 isms under the num~er DSM 23'~7 and appropria~e plasmids~
having the same repliçon are partiçularly preferred.
~7~3~
The pLasmid w;th selec~ion markers which is em^-
ployed in process s-;ep b) can be the plasmid pBR 3~2
which is described in Gene 2 (1977) ~5-1130 The hybrid
plasmids ~hus ob~ained are denoted pRMX in the tex~
5 which follows n
A hybrid plasmid which contains the genetic infor--
mat;on for ~he expression of insulin, as is dPscribed in
European Pa~ent Application 0,032,675, is also suic~
able as a plasmid ~ith select;on markers whic17 can be
employ2d in process scep b)~
The hybrid plasmid ob~tained by process step b)
is then introduced into a suitable host or(~anism,
advan~ta~eously Escherichia coli, by ~transforma-
~;on, and ampl;fied there.
In process step d) a su;cable conju~ative plas~
m;d~ advanta~eously RP 4, ;s ;ntroduced ;nto this host
organ;sm which conca;ns the hybr;d plasm;d. In addi
t;on, .he mob;l;zabil;ty defect is complemer1ted by ;n~
troduct;on of sui1ta~le plasmids, such as Col K and Col V0
The host organism thus prepared can then~in pro~
cess step e), transfer by conjugation the hybrid plas-
mid in~roduced in process s~ep c) ~to a,preferably plas-
rr,;d~free, obligate metl1ylotrophic bacter;un as reci-
pient~ Preferred rec;p;ents are barcer;a ~f ~he ~enus
Me~hylomonas, in particular of the species Me~hylomonas
clara, abcve all of the s~rain ATCC 31 ~26. This strain
is described~ for example, in ~erman Patent 2,~33,451
and US Pa~en~ b,166,D~
- Firlally, in process s~ep f~ ~he desired clones
~ - 4 -
are selected by culturing in z medium which contains
methanol as the carbon source and on ~he basis of ~he
transferred resistance or sensitivity ~owards antibio-
~ics. The obliga~e methylotrophic bac.eria thus ob-
tained are capable of expressing ~he con~ained foreign~NA, and thus, for example, of producing insulin.
De~ails of these process steps are expanded on
uithin ~he framework of ~he examples.
Furthermore~ the invention relates to a varian~
of the above~,entioned process, in which the process
steps d) and e) are simplified as follows:
After selection, in s~ep d), the hybrid plasmid is isola~
ted and, in step e), the isolated plasmid is introduced
by ~ransformation into spheroplasts of the obligate me-
thylotroph;c bacter;um.
Thus the ;nvent;on also relates to spheroplastsof methylotrophic bacteria, particularly of obligate me-
thylotrophic bacter;a~ especially of bacteria of the
~enus Methylomor,as, preferably the species Methylomonas
clara. The stra;n Methylomonas clara ATCC 3~2~6 is par~
ticularly preferred.
The spheroplasts according to ~he invention can
be prepared by culturing the bac~eria in a glycine~ric~
medium con~aining an osmot;c stab;lizerO Th;s med;um
is preferably sligh~ly hypo~on;cO Fur~her preferred em~
bodimen~-s of this preparation process are explained in
more detail below.
Furthermore~ ~he ;nvention rela~es ~o the use
of ~he ne~ spheroplas~s for inser~iny ex~ternal DN~ in-~o
" ~7~L6
~. 5 ~
these bac~eria. This external Dr~A is pri~aril~ in~ o-
duced in the form o~ a plasmid. Suitable plasmids are
the abovemen~ioned hybrid plasmids.
In order to produce ~he spheroplasts, ~he methy
lotrophic bacteria are cultured in a sui~able medium,
advantageously in a minimum me~ium, up to a suitable
cell densi~yD Sui~able cell densities are in ~he OD600
range of r preferably, 0.5 to 1 ~, in par~icular 0.9 ~o
1.4. A defined volume of ~hese cul~ures is then in~ro-
duced into about S ~imes the amoun~ of the m*dium~entloned, which is morec~ver ~lycine-rlch and conl:ains
an osmot;c stabll;zer. The con~ent of glyc;ne can be
as much as the saturat;on concentra~;on~ a content of 2
to 4~ by we;ght be;n~ pre~erred.
Su;~able osn~ot;c stab;lizers are sugars, such
as sucrose, sugar base alcohols, such as sorb;tol, and
polyglycols, such as polyethylene ~lycol 6000. The me-
dium 1s preferably slightly hypotonic, this being
brought about by suitable concentra~ions o~ the osm~ic
s~ab;l;zer. ror example, a sucrose concen~ration of 10X
by weigh~ or 1 mol~r sorb;~ol is suitableO
~ he bacter;a are shaken in this medium un~il es-
sen~;ally no rod~shaped me~hylo~rophic bac~eria are de-
tec~able under the phase con~rast m;croscope~ In gene-
ral, after about 1 hour of shaking (100 to 1gO rpm) atabou~ 37~, the produc~ion of the spheroplas~s as im-
mobile spherical structures can be observedO rhis pro-
cess is generally comple~e 3f~er abouc 4 hours~
The produred spheroplas~s are carefully
~7~
-- 6 ~
cen~rifuged c'own, for example at 2600 x g and 4C fcr
10 minu~es, and resuspendeci in su;table med;a. rhis re-
suspension mediurn likew;se contains an osmo~ic stabili-
zer at 3 sulta~le concentration, for example 15% by
S weigh~ of polye~hylene glycol of rnolecular ~eight 6000,
to stabilize the spheroplas~s.
A mixture which comprises equal par~s Qf the
rnedium used to resuspel-d the spheroplasts and the car~
rier for the DNA to be introduced~ in a sui~able buf,err
1D is added to this suspension at an approximate volu~e
ratio of 1:5 D A suitable DNA carrier is, as detailed
above, primarily a hybrid plasm;d~ A buffer suitable for
this purpose comprises an aqueous solu~ion containing
10 mmol of TRIS (tr;s~hydroxymethyl)aminomethane) and
1 mmol of sodium e~hylenediaminetetraacetate ~TE bu~
fer) per liter. An osmotic stabilizer is added to ~his
mixture tfor example 4~ by weight of polyethylene glycol
6000 solut;onr three t;mes the volume oF the suspenslon
of spheroplasts) ana it ;s carefully and ~horoughly
mixed. After standing briefly at room te~pera~ure~ the
resuspension medium, about ten times the volume of ~he
suspension of spheroplas~s, is added and the mixture i5
carefully centrifuged ~3600 x 9).
1he precipitate is then taken up in resuspension
med;urn, twice the volume of the suspension of sphero-
plas~s, and s~reaked on~o agar plates to check ~he~her
transfor~a~ion nas ~aken place. These aaar pla.es have
a composition correspond;ng to the resuspens1On me~iurn
and con~ain, in addi~ion to 1~5~ by weiyh~ of Bac~o agar,
-- 7 --
an arlt;ob;o~;c suit~ble for selec~ion, for example 5C IAg
/rnl of ampicillin or 10 ~g/ml of tetracycl;ne, in tl-e case
where ~he hybrid pla~ids employed contain~d the approp-
r;ate resis~ance genes~ The plates are ~hen incubated
a~ 37C for 1 to 3 days, and resis~ant colonies~ af~er
being cultured in ~hç abovemen~ioned liquid medium wh1ch
contains 2 suitable antibio~ic, are investi~ated for ~he
presence of plasmid DNA (Humphreys e~ al~, BBA 383
(1975) 457-463). By th;s means, the plasmids ernployed
10 for transforma~;ion can be isola~ed from the me~hylotro-
phic bac~eria.
~ ur~hermore, the invention relates ~o the plas-
mids from the Mer.hylomonas clara strain DSM 2397 and the
hybrid plasrnids with a replicon inherent to an obligate
r,~thylo~rophic ba~ter;um, jM other words plas~i~s ~Ih,~
are repl;cated ;ll the bac~eria of the ~enus 'Methylomo-
nas~ preFerably of the species ~lethylomonas clara, in
part;cular the stra;n ~TCC 312~6, ;n ~h;ch prokaryotic
or ~ukaryotic DN~ is in~egra~ed, in particular ~hat for
the express;on of insulln~
Fur~hermore, ~he inven~ion rela~es ~o ~he host
organisms which con~ain the pLasmids mentioned, and ~he
hos~ organisms which addi~ionally con~ain the conju~a-
~ive plasmid and~ furthermore, those which con~ain addi-
~ional plasmids for the aboli~ion of the mobilizationdefectD Pre-ferred host organisms contain RP 4 as ~he
conju~ative plasmid and Col K or Col V as plasmids for
~he a~olition of ~he mobili~a~iorl de^fec~
The acJvan~age of the invention comprises use
'
2~6
- 8 -
be;ng made of a replicon of ~ plas~id fro~n an obliQOte
me~hylotroph;c bac~erium, in other words one ~i.h a very
r~arrow ran~é of hosts Thus the process according to
the invent1On is distinguished by a high degree o~ safe-
ty, so that it can be applie~ ~o recorlbinant bN~ which
contains hazardous information.
~ he invention is illustrated in more de~ail in
the following examples. In these, percentage da~a re-
late to weight unless otherw;se specified~
In th~ examples, the following abbreviations are used:
ATP = adenosine triphosphate
EDTA ~ ~thylenediarn;netetraacetic acid or -acetate
tNa)
OD6~0 = optical density at 600 nm
TE-buffer = aqueous solution containing 10 mmol of tris
HCl per l;ter, adjusted ~o pH 8, and 1 mmol
o~ EDTA per liter, also adjusted to pH 8.0
Tris ~HCl)- trishydroxymethylam;nomethane ~hydrochlo-
r;~e).
In the Drawin~s
Fiqure 1 shows a restriction chart of plasmid
pBE 3 isolated from the strain Methylomonas clara
DSM 2397; and
.~ .
Figures 2 and 3 show hybrid plasrnids containing
the complete DNA of plasmid pBE 3 and DNA from plasmid
pBR 322, the latter being inserted in both of the
possible directions.
- 8a
Examples
1. Isolation of plasmid from M. clara DSM 2397
The plasmid was isolated essentially according
to the method of Humphreys et al. (BBA 383 457-463).
Fox this purpose, the bacteria from 1 liter of culture
medium having an OD600 of about 1.0 were centrifuged
down and the precipitate of bacteria was resuspended
in 5 ml of sucrose solution (25% sucrose in 50 mmol/l
of tris HCl solution of pH 8.0). While cooling in ice,
1 ml of lysozyme solution (5 mg/ml of lysozyme in 250
76~
9 _
rnmol/l of ~ris I~Cl s~lu~ion~ p~ &~Oj and 2 ml o~
0~2 molar EDT~ solu~ion of p~l 8.0 ~ere ad~ed. ~he mix-
~ure wa~ incuba~e~ on ice ~or 5 r~inutes with occasional
swirlin~ Lysis was ~hen brough~ abou~ by the addi~ion
S of 8 ml of a mix~ure con.ain;ng S0 mmol/l of cri
H~l, 75O5 mrnol/L of EDT~ and 0.2% of 2 nOn-iOlliC
surFac~ar)t ((R)Tricon X~100) of pH ~Ø The highly
viscous mixture was centrifuged at ~8~0D0 x ~ for 3~ rni~
nu~es, a clear lysa~e being obtained as the supernatant.
10 Per 1D rnl o-F supernatan~, 101 ml of 5 molar saline and 1~1
9 o~ polyethylene glycol, of meall molecular weight ~,Oi30,
were added. Tlle m;xture was incubated overnight at 4~
The flocculent precipitate was collected by centr;fuga-
tion at 1500 x ~ for 5 minutes and d;ssolved in 3.~ ml
1S o~ buFfer solu~cion tS0 mmol/l of tris HCl~ 5 mmol/l
of NaCl, pH 8~0), and the volume of the solution
was measured. Af~er addition of 1 9 of CsCl per rnl of
solut;on and of 1/15 ml of a 1% strength aqueous ethi
d;LIm bronl;de solution~ centrifugation uas carried out
for 10 rninu~es at 16,0ûO x g. The supernatant frorn ~chis
was cen~rifuged to equilibr;um (~0 hours at 47,090 .revo-
lut,ons per rnîllu~e, 18C, in a Yertical rotor) and
showed ~r~o bands which were clearly visible, even ~i~h_
ou~c irra~iation ~ith U~ ligh~c, lying abou~c one centime-
~er apar~c. rhe ~luorescen~ bands were accentuated byirradiation with UV light of wavelength 366 nm~ and the
lower pl.asmid band was harves~ed by piercing che gra-
dien~ 1rorrl ~he side ~ith a needle and subsequen.ly aspi--
ratin~l ~he barld ;n a syringe. rhe e~hid;um bromiàe was
z~z~
10 - -
removed by repeated extrac~ion by shaking with i50pr~pa~
nol sa~ura~ed wi~h CsCl. Af~er dialysis a0ainst 2
liters o~ TE buffer 3 tiloes~ for at leas~ ~ hours
each time, ~he plasrnid DNA was ob~ained in a puri~y
5 ~h;ch permitted i~s use in the folluwing prccess steps.
I~ impurities were s~ill eviden~ in ~he DNA ~o-
lucion, i~ was excracted twice with iden~ical volumes of
phenol sa~ura~ed wi~h 0.1 rnolar ~ris HCl solu~ion of pl'
8.0 and then 3 ~imes with absolute e~her~ The excess
e~her was blown off. 1/~ of the volume of ~hree rnolar
sod;um acetate solution was added to ~he solution, and
the DNA was precipi~ca~ed by add;~ion of the same volume
of ;sopropanol~ After standing overnigh~, the mixture
was centrifu~ed a~ 12~000 x ~ fùr 30 minu~es~ and the
DNA precip;~ate was washed w1th ~0X s~rength ethanol.
Subseque~tly, the PNA ~as freeze~dr;ed and .a~en up ;n
TE buf~er.
2. Isolation of DNA froln ac~arose
In ~cl~e process which follows, the method descri~ed
by Lan~ridge et al. (Analytical Biochemistry 103 ~1980)
264-271) was used.
The DNA was applied ~o a horizon~cal 0.5% strengtl
agarose ~el (low~melting a~arose, ~ype VII, NoO A-~018,
S;~rna). hfter elec~rophoresis, the bands were visualized
by s~aininy wi~h e~hidiurll bromide and cu~ ou~ of ~he gel.
~he agarose disl con~aining ~he D~A was melted in a glass
~ube a~ 70C~ and the volume was measured, and it was
cooled CIGwn ~o 37~C. To ~his were added es1ual volu~es
-~ o~ the bu~anol and wa~er phases described below.
- ~.;27~
1 1 ^
150 ml of n~bucanol were shaken with 150 n~l of
wa~er in a separating funnel,and~ af~er separ&tioR OT
the phases, 1 ~ of hexadecyltrinletilyLalllMon;um bromide
was dissolved in 100 ml of the water~saturated butanol
S phasea This solucion was ex~racted by shak1ns with 100
ml o~ the wacer phase, i~ being possible f~r a defoamer
to be added (50 ~1 of An~ifoam A, Sigma). After separa~
tion of ~he phases overnight~ they were collected sepa-
rately.
1~ The mix~ure obta1ned af~er addi~ion of these bu~
tanol and water phases was thoroughly mixed by carefully
rotating the tube, and the phases were allowed to sepa-
rate at 37C. The uppe~r butanol phase con~ain;ns ~he
DNA was separa~ed off, and the remaining aqueous phase
1~ was extracted twice more w;~h bu~anol in the manner des~
cribe~ One quar~er of the volume of 0.2 molar NaCl
solut;on was added to ~he combined butanol phases, and
thorough m;xing was again carried out. Af~er ~he aqueous
phase had been separa~ed o~f, another salt ex~raction was
2n carried out and ~he same volume of chloroform (which had
been purified over an aluminu~ oxide column) ~as added
dropwise to ~che combined aqueous phases~ After standin~
on ice for half an hour, the lower chloroform phase was
discarded and ~he remain1ng chloroform ~!as blown ou~
wi~h air. The DNA was precipitated wi~h isopropanol and,
af~er separat;ng off~ resuspended in TE buffer.
3. El~ p~ si_ o__E~ a _ yla~,ide~
8 ~ s~crength polyacrylamide (PhA~ ~3els ~Jere
used for ~he visual;~acion and character;za.10n of DNA
2~i
~ 12 -
fragmen~s below 093 MD. The sels were 1 mm thick, 3~ cr,
long and 14 cm wide. A mix~ure contatning ~9 rnrnol oF
boric acid~ ~ mmol o^f tris HCl and 2.5 mrnol o~f EDTA
per L1~er and haviny a pH of 802~ served as ~he buffer.
S In order to prepare an 8~ PAA ~el, 33 ~l of a
24% s~rength PAA stock solu~ion t23.Z2 9 of acrylamide
and 0.78 9 of ~ methylenebisacrylamide in 100 g of
aqueous solut;on), 10 ml of the electrc)phoresis buffer
men~ioned, ~hich however con~ained aLL componen~s in
1n 10-fold concentr2~ion~ 6.25 mL of a 6~/t% s~reng~h 3~
ethylaminopropioni~ri Le solution and 50.75 ml o~ wa~er
~ere mixed. The mixture was degased on a ~a~er pump, and
polymerization was started wi~k solid ammonium peroxodi
sulfate. The gel was poured immediately thereaf~er, and
a m;nimum oF 2 hours was allowed to elapse before the
start of elec~rophoresis. Before apply;ng ~he samples,
a voltage of 100 V was applied for about one hour ~o
the gel to rernove resi~ual ammonium peroxodisulFate.
The eLectrophoreses were carr;ed ou~ a~ 100 V
and a current of 12 mA.
4. C_aracterizat on of ~h~lasmid ~BE 3 from the
in ~S~ 23~7
The plasmid pBE 3 was digested wi~h ~he re~
str;ction endonucleases men~ioned in Table 1, and the
~5 fragments produced were separated by geL electrophore
\~ SiSn The results recorded in Table 2 ancl Figure ~ ~ere
ob~ained by double diges~ions and by charac~erizat;on o~
;ndividual fragmen~s cloned in pE~ 322~ as described
below.
%7~21~
~ 13 o
- The plasmid pBE 3 is a dele~ion mu~c-7n~ o~ a
larger plasmid~ Besides ~hisr smaller plasmids also
exist. All ~hese plasrl7ids are equivalen~ in ~he sense
oF ~his inven~ion when ~hey con~ain the replicon inhe~ ¦
S rent to Methylolnor7as.
rah le 1: ~
Survey of the enzymes used for ~he charac~eriza~ion o~ ¦
pBE 3:
Enzyme Num~r oF ~ragment~ I
10 Acc I
Xor II
Eco RI
H;nc II S
Ava I 12
15 ~al I a~ leas~
No cleav3ge with
~am HI
P9l II
~0 E3st EII
Eco RV
Hind IXI
l1pa I
~pn I
Nru I
Pst I
Pvu II
Sal I
Sma I
gLZ~Z~6
~ 14 -
- Table 1 ~continued)
Sph I
Sst I
~st II
Stu I
Xba I
Xho I
X~n I
Table 2:
__ _~_
Size o~ the frag~ents ~;n MD) wh;ch were produced by
cleavage of p~E 3 wî~h restriction nucleases:
EcoR I: H;nc XI- Ava X:
F1:4.14 H1 : 6.8 A1: 2.0
1 5 F2 :3 ~ 09 H2 : 1 . 42 A2 : 1 ~ 72
F3 :1 .27 H3 : 0 . 87 h3 : 1 .26
F4 ~1 H4 : 0.31 A4a: 1003
FS:0.63 H5 : 0.28 A4: 1.03
A5: 0.85
~ A6 : 0 . 66
A7: 0.55
A8: 0.50
A9: 0.13
A1 0: 0~08
A11: 0 ~ 07
EcoR I~ EcoR I~
hva I: Hinc II:
D1 : 1 r35 E1 1 2 . Z9
D2 : 1, Z2 E2 : 1 ~ 83
- ~2~2~6
15 -
Table 2 (con~inued)
D3a: 1.03 E3 : 1~42
D3 : 1.0-~ E4 : 1.Z7
D4 : 0O85 E5 : ~).91
D~ : 0.70 E6 : 0.63
D6 : 0.63 E7 : 0.~6
D7 . 0~55 E8 : O./tO
D8 : 0.5~ E9 : 0.31
b~ : ODS3 E10: 0O2
D10O 0~50
D11: 0~4
D12: 0.1~
D13: 0~12
D14: 0~1
: O.i
D16: 0.1
S. Res~ric~ion diges~;ons
'Cnd;vidual diges~;ons were carr;ed ou~ in a ~o~
tal volume oF 50 to 100 ~l with ~he hufFers recommended
by ~he manufac~urers. In order ~o ensure complete cii
' gestion o; the DNA, ~he samples were incu~ateci over~
n;gh~.
For double and multiple ciiges~ions~ ~he approp~
2.5 ria~e enzymes ~ere usually adcied ~oge~her ~o ~he DNA.
In these cases, ~hP buffer comprised a solution contain~
;ng 50 mmol o~ sodium chloride, 5 mmol of ~ris HCl
(ad~us~ed to p~ 7.5~ 6 mmol of ~agnesh!m chLoride, 6
mmol of 2~mercap~oethânol and 100 rn~ of bovine serum
2~2~i
~ 1b
albumin per liter. Control experirnents showed that ~he
enzymes provioe the same resul~s in this buffer as in
those recolnmended by the rnanufacturers. The exceptions
were enzymes whi~h have an above average salt re~uire~
men~ In these cases, diges~ion was initially carriecl
out at a low salt concen~ration wi~h one enzyme~ and
the second enzyme was only added after increasing the
salt conc E n~ration~
6~ Par~ic~l d;ges~ions wi~h Eco R I
2~ 9 of pLasmid DNA were in~ubated wi~h 1 U of
the restric~ion enzyme a~ 37C in a ~o~al volume of
50 ~Al. Identical ~;xtures were incubated for differen~
times, and the reaction was s~opped at ~he various times
hy heat;n~ the rnixture at 70C ~or ten minutes. Ana~
lysis of the par~icular pa~tern of bands by 0el electro
phoresis showed the.extent to which ~he digest;on had
advanced after the defined t;mes.
7. ~row~k ronditlons
hll Escherichia coli s~ra;ns used were cultured
in L broth ~10 9 of Bactro Trypton, 5 g o~ yeast e~rac~
and 5 Q of sodium chloride per 1 liter of water)~ The
Me~hylomonas clara strains were cultured in one of the
~wo followlng minimum med-;a:
3~ 5% of me~hanol
0~1% of H~P04
0l0~3% o~ ~S04
O~D1~X of Na2S0~ H~0
0~3S% o~ ~S~4 O 7 ~l2
-0 004JO of CaC03
`" ~2702 G
~ ~,
O.O~S~ of citric acid
0,0~5% of (NH~t)2Fe(S04)2 6 H20
~o~ l of t,ace elemel1t solw~ion
V 135 L D 1 L5% of methanol
n.16~ of K2S04
ODO6% of MgS04 . 7 H20
tl.O2S~ Of Na2SO4
0.014% Of GaCO3
Q~01~ of F~2(S~4)3
~0 0.2~ of ~13POf~
0.28% of NH3 (25%)
0.3YO of KN03
0.3X of NaHC03
. 1 mL/l of ~race element solution
Trace element sol(~ion:
0.05 g/l of H~B0
0~01 g/l of KI
0~04 g~l of MnS04 . 4 t~20
0.~4 ~/l o~ Z~S04 . 7 ~
n.o2 g~l of (NH~)6Mo7024
. Construc~ion of ~he hybrld vectors
The plasm;d pBE 3 was partially d19es~ed with
the restriction enzyme Eco R I so tha~ ~he major part
of the plasmid ~as only cut once and was presen~ in ~he
linear form. pBR 322 DNA was comple~ely digested with
Eco R I and ~hen subjected to treatmen~ with alkaline
phosphatase. The DNAs thus prepared ~ere mixed and in~
cubated~wi~h T4 DN.~. ligase at 14C overnightD S'J~ of
~his liqa~ed mixture then served for transformation ;ntO
1~7~
18
~he E. coli s~rain ~l~ 101 made competen~ by treatmen~
w;th CaC l2 .
The bac~eria were smeared onto L broth pla~es
with 20~/ml of tetracycline and incuba~ed overni~htr
5 Resistant colonies were reinoculated on fresh pla,es and
well-gro~n clones were inves~igated for ~he presence of
plasmid DNA having a higher molecul~r weight ~han p~R
322 by "single colony lysis"~ Clones !Jhich con~ained
plasmid DNA of ~his type were ~inally cul~ured in 1Q0 ml
of L broth wi~h or without tetracyclire~ and, after chLo~
ramphen;col stimula~ion of ~he bac~eria, ~he plasmi~ DNA
was o~tained.
Res.riction diges~ions with the enzymes Eco R I
and Ava I showed ~hat ~he hybrid plasrnid pRM 21 shown
in Flgure 2 and the hybrid plasmid p~M ;4 sno~n ;n Fi-
~ure 3 had been obtained.
In addition, it is poss;ble ~o identi~y hybrid
plasm;~s containin~ Fractions of pBE 3.
9. Lic~as~ reac~ion
_ _ __ _
L;gase reac~ions were carried ou~ in a volume of
50~ l at a total ~Nh concentrat;on of 20 ~g/rnl. 1he
buffer contained 30 mmol o~ ~ris HCl (adjusted to pH
7~5), 4 mmol of ma~nesiurn chloride, lO mmol of dithio-
ery~hri~ol and 0.2 mrnol of hrp per liter. 1~l o~ T~
2S li~ase~ correspondir,g 'LO 400 Ur was added to ~hese rnix
tures tin this contex~, 1 U corresponds ~o the amount
of enzyme necessary ~o liga~e 50% o~ lambda D~!A, irhich
has been di~ested with H;nd III, ;n 30 minutes a~ ~16~C
in a volume o~ ~O~l. T~e concentra~ion of ~ e ~NA ir,
~7~z~
this mixture is abou~ 330 ~g/ml~. The ratio of the ~o
DNhs to one ano~her which is op~imum for a par~icular
ligase experimen~ was calculated by the method of Du-
gaiczyk e~ al~, JMB 96 ~1~75) 1 11 . The incubation was
S carried ou~ a. 14C for at least 16 hours.
10. Con~ju~tlon
Recipient and donor were cultured overni~h~ to
give an OD6~D of 1nO ~ 1r~ (recip;ent) and 1.4 o 1~6
f tdonor)~ and equal volumes of recipient and donor were
10 mixed so ~ha~ the mixture had a large available surface
area~ The mix~ure was incuba~ed, w;~hou~ shakin~, a~
37C for ~ hours, and then 20C )~l of it was ~meared c~n- ~
to ayar pla~es, the compos;~ion of wh;ch permitted se~ P
lection a~ainst the donor and For ~he receptor, ~he
lat~er bein~J provided with a new prop~r~y ~!hich ~!3S
supplied by the plasrn;d to be transferred. For the
15 conju~a~;on between E. coli HB 1D1 and M. clara DSM
2~7, these were methanol-minimum m~dium plates wi~h
the add;t;on of arl antibio~;c tdetermined by the type
of the plasnl;d to be transferred)~
In o~her experiments, recipient and donor uere
;ncubated as described above and 2Qû ~ l of this mixture
was smeared on~o me~hanol~minimum medium pLates wi~hout
anti~io~ic. These plates were incubated at 37C over- i
.5 night, and the lawn of bac~eria produced was rinsed off
~ith 2 ml Gf methanol~minimum medium~ 200 ~l of this
suspension ~ias then again smeared on me~hanvl~minimum 1-
medium plates wi~h the appropria~e an~ibio~ic~ Clones
emerged after incubation of the plates at ~7C for r
I
, .
- ~.2
- - za
48~72 hours.
11. Expressicn o~ o~i~ DNA in M. clara
a) The pLasnlid pBE 3 ~Jas cloned or recloned
by the processes described in derivatives of pBR 3~2
which con~ained~ as ~he c~DNA sequence o~ a eukciryo~ic
~ene~ that of monkey insulin.
The monkey insulin c-DNA had been incorpora~ed
;n the Pst I cleavins site of pBR 322 so that this
forei0n informa~ion was expressed under ~he con~rol or
the ~-lactamase prornotor in E~ coli~ 1h;s produced 2
fusion protein which could be detec~ed with anti insulin
an~ibodies ~European Paten~ Applica~ion 0~032J67~.
Monkey insulin c~DNA contains two in~ernal Pst I
cleaving sitesu Thus the coding infornation cannot be
removed in an intact forln from ~he plasmid by Ps~ I di
gestion. Thus in order ~o make possible replication of
th;s plasmid ;n M. clara, the procedure was such tha~
DNA sequences from the M~ clara plasmid pBE 3 ~lere in-
corporated in this pBR :~22 derivative expressing insu-
~0 l;n information~ The experimen~al procedure was as des-
cribed above. By this means, a number o~ hybrid plas-
mids were ob~ained which differ ;n their struc~ure frorn
the hybrid vec~ors described above onLy by the Inon~
key insulin c~DNA incorpora~ed in ~he pBR 32~ par~
The arrangement of thc insulin gene in ~he pBR 322 part
of ~hese vec~ors s~ill remains in phase, so ~ha~ ~hese
clones also express insu!in~a~1ti~3enic deter~linan~s in
coliO One of ~hese clones, wl-;ch contairls ~he n~ire
pBE 3 sequel1ce~ ~as given ~he name pInMc 6~.
21 ~
b~ The rmobilizability defect foul)~ in pB~ 372
can be complemen~ed by plasmids such as CnL K and Col
(Youn~ and Poulis~ Gene 4 (1978) 175~17~). Thus, in
addi~ion, the plasmi~ Col K was in~roduced by trans~
forma~ion into those donor strains ~i~t) the conju~
ga~ive plasm-id RP ~ and ~he hybrid vec~ors o~ the p~M
series which had been rnade competent by trea~ment
with calcium chlor1de and are described above. The
colicin~ensi~ive strain AB 1157 was used as the in~
1D dicator strain to detect ~he plasmid Col l~ (~arren e.
al., MGG 170 (1~79) 1D3-107)~ In experin1ei1ts 0l1 cor~Ju
gation be~ween the En coli donor s~rains HB 1~1 (RP 4,
Col K~ pRM 5~) or HB 101 (RP 4, C~l K, pRM 21) and
~he recipien~ M. clara ATCC 31226~ in order to select ~Of`
clones which contained ~he hybrid ~ector, selection was
carriecl ou~t in the presence of high doses of tetracycline
~50 ~g/nll) on me~hanol~mil1irnum n~ed;uln (M 36).
Analys;s of a larye num~er of Mu clara clones
showe~ ~hat, in about 10% of ~he case~ clones had be.en
obtained which only containod the hybrid vec~or~ :[n- an
experirilert on conju0atiorl be~f~eerl the E. coli donor
s~rain l-IB 101 (RP 4, Col Kf~ pInMc ~8) and the M. clara
recipien~ ATCC 31226, in the salne manner~ M. clara
clones were ob~a1ned whicl1 only con~ained the plas
m;d pInMc 68.
c~ rhe M. clara clotles wi~h ~he plasmid ~InMc
6S obtain~d ~lere ~hen cultured an~ checked for their in~
sulin conten~ by a radioimmunoassay or a fat-c~ll assay~
Consis~en~ wi~h ~he observa~ior!s made on ~. coli in ~he
- 22 ~
~t,'~ European Pa~en~ Application 0,032~675, insulin values
be~ween 1 and S IU per liter were also measured in the
case o~ M. clara ATCC 312260 Thus the insulin informa-
tion contained in ~he pBR 322 par~ of ,he hybrid vector
S pInMc 68 is also correc~Lly and ef~ic;ently expressed
in M. clarar
12. The s~rain ~,ethylomonas clara ATC~ 31226 is cul-
tured in the min-,mum medium M 36 ~o an OD~oo of 0.9 ~o 1.4.
20 ml o~ ~hese cul~ures are introduced in~o 100 ml of
M 36 r~edium whicll additionally contains ~% glycine and
10% sucroseO The bacteria are shaken at 100 ~o 180 rpm
in this Medium at 37C for 4 hours. No oblong mobile
bacteria are ~hen any longer detectable under ~he phase
contrast microscope.
1~ Thc spheroplasts produced are cen~ri~-uged down
a~ Z~00 x 9 and 4C for 10 minutes and resuspend~d in
1 ml of M 36 medium which additionally contains 10% suc-
rose.
The media mentioned can also conta;n, in pla~e
of 10~ sucrose, 1 mole per liter of sorbitol or 15%
polyethylene glycol of mean molecular weight 6000.
The spheroplasts can also be separated out by
cen~rifu~a~ion at 3~00 x 9 and 4C for 10 m;nu~es.
13a 0~1 ml 0~ a mixture comprising equ~l par~s of
resuspension medium and TE buffer and con~aining the
plas~id pRM ~1 is added to 0O5 ml of the suspension ob~
tained as in E~ampla 12. 1.5 ml of 4% streng~h poly-
~llylene glycol 600D solu~ion 1s added to this mix~ure
~nd i~ ;s carefully and thoYoughly mixed~ rhis mix~lJre
z~~ --
i5 allo~ed to s~and a~ room tempera~ure for ~ m;nutes~
5 ml of resuspensior1 medium are added and it is centrl--
fuged al 3~00 x 9~
The precipitat~ is taken up in 1 ml of resuspen-
S sion rnedium and s~reaked on agar pla.es, the compositiol~o~ which csrresponds to the resuspension medium an~,which a~di-
tionally con~ain 105% Bacto agar and S0 ~ g/ml of ampi-
c;llinD The pla~es are incubated at 37C for 3 days~
and the colonies are cul~ured in M 36 rnediurn which con
tains S0~9/ml o~ ampicillin. It ~as possible ~o isolate
~he plasmid p~M 21 employed for transforma~ion from
~the bacteria ~hus obtainedn
It is also possible to employ in this process
tetracycline at a concentra~ion of 10 ~g/ml in place o~f
1 5 aMpi ci llin.
The same result ;s also ob~a;ned ~hen, in place
of the abovelllent;on~!d plasm;d, the plasmid pRM 54 is
employed~
14. If the proce~ure is carried ou~ as in Example
13~ but ~he plasmid pInMc 68 is employed and selec~ion
is hy means of 50 ~g/ml of ~e~racycline, then Methylo~o-
nas clara clones l1aving this plasmid and producing insu~
lin are obtainedn
