STABILIZATICl\I IF EXFf£SSICl\I CF l¥'CILLUS TH.RII\I3JEN3.1§
9JES='. ISF:PEL..ENS!S IN ESD-ERIO-HA CCLI I'4\ID CY!'NJBPCTERIA
lJTILIZII\I3 n-E F'I'f':B UD..JS CF Ft..A2MID Pi.
BY
s-£F.RY L.
~
ADVISCF: - DR. CPf\11..YN VPN\I
PPU... STATE LNIYERSITY
I"I..N:IE,
IM)I~
Abstract
The proposed study is ccnc:enled with the stabiliz'rtio'l of expressicn of
to><in genes in ESC:D:;>Li&:j:li;}
£:.9_ti and HE cyanobacterium
Syn~h::!cc£,~us
Fa::: 7942
.Jhich are speci Hc for tt"E larvae of t!illRheles 9b!§drimachlJ.;;>tL!,§; rrosqui tees which
may carTY hJflk:\f1 malar'ia.
~c:.iJJ}::§ tt~lC:~tlQ.:t~?i§.
Tre to}-:in
gB1E.~:r:3
ar-e obtainE?d fran th? soi 1 bacter'ium
subsp_ :h§!:.ge1eps:h§: (B.t.i.) ~-..jhich pr-oduces a c:ry~-:)tall.ine
endotm:in canpo'"""'CI of insecticidal polYpE'pticIEs.
Clones have been construct.<ed
which cont,,,in pelr"t crf the tm:i" gem? in the hyb,-id vector- pTNTV, which is able
to transfDrm both
s....
col,i and c:yanobac:ter'ia.
Twelve of these clc:n€."S produc:,,,j
signi ficant tm:,icity to fc:urth instar larvae o'f a more SLI,,ceptible mc)squito
spE'Cies Aec:LiE Aeqypti.
T,,,', of t.t"-Ese
t.welv'~
clc:nes have re<::er",Uy been shew", to
have lost. tt-eir !:l.",i • .i. inse,-t due t.o seqr€.."9at.iolal instability of the plasmid
duF'ing ,-epl icat.ion.
Tt"E plasmid p<Gl022 cc:ntains a @r::!:l. locus encoding
a
plasmid st.abilizatic:n syst€.."fJl whieh has been she"""" to st.ab.ilize 1;,..", Q21ic_ and
other gram negative bacteria.
The pr'imary goal of t.he proposed research is t.o
inser't t.his P,:w!:l. locus into the plil,smid DI\IC1 of the vector pTNTV and the clO'le5
.,hieh st.ill show t.m:icity, and t.o det.ermine if t.his f!9cI! locus st.abilizes e>:--
pressicn of the to}(in.
Malar-ia is a disease which t.hre"t.ens many regicns of t.he world.
b-ansmi tted v_i" the bi t.e crt t.h., eoQP..hel<;§ lrosqui t.o; the,-e-fore,
vec:tOt- of
malar'ia~
the disease.
tt-e
f.:l1qpJ.'@.i~? mc.-,squito~
i~~)
t.a'-get1.ng t.he
of great imp::Jf'tance in limiting
0..,,- "pprO<lch has lea,.., to c lcne the rrosqui t.=id"l t.o>'_in of
';;:~_b!?Fis.t>i"
sequent.ly into cyanobacteria as a lT€ans to centro! 1Ik3laria.
clcnes which c:cnt.ained high levels of to>:icit.y we,-e ident.ified.
~
'''''5
_isolated fn:lI1l t.t-.., t.o>:ic: c: lcnes.
that elenes prcxJueed t.hra_lgh
!:::s.~I
~k3d
lost. there
p-NXI0 and .H"X3 st.ill eont"ined the plasmid insert..
clene pl-NX.10 gave
SE..:eJl
LIS
under' t.h:--? phase microscope, and it.
The plasmid
[N!.i
j,S
[N!.i
sh<:Mled
insert.,
lili?_I
lig"t.ic.TI,
Furt.her analysis of the
t.wo addi ticnal pieces of informaticxl.
to Aedis il<=qypti_ 1"rvae •
~~,J;.,j.-,_
elenE'S pn::duced thn:xJgh
coJ.A
The plasmid
Pnal ysis of the pl"smid
lig"tien
SltlJgesting segrE<Jaticnal inst."bility.
crystal as
It: is
It. produced "
still pn:x:lucing to}:ic:i ty
fran p-NX10 wi 11 be used to t.ransfol'"m
P-lt into cyanobact.eria t.he delta-g1dCJt.0>,.in is nCJt. e,-,pres",ed as well as in \;....
coli (1.)
The lli:I.rE' l=us provides" way t.o en!5Lll'"e pl"smid st.abilizaticn (8)
and is h:Jped t.o incn,ase t.he ii\ITlC<_lf1t of to>:in produced in both
cyanobac:t.er-ia.
EO...._ galA and
F"€VIEW CF LlTERATI..F£
Me.-·\!i:H'""ia is a wide-·--spr-ead
time having tre disease.
E~pidE:"l1lic
\.'-Jith 100 mi 11 ic:n pec)ple at. a.ny gJ.\lefl
Of tt--esr" 100 millicn people, anJund cne million--
mostl y very young chUdn",-, in
~)fr-ic:a--,Jie
fr-om malar-ia each year- (11.)
In the
tropics the ef-fects ar-e so wide--spr-ead that malaria has been blc'llTlE'd for- impeci-ing tre developnent of entire natiens (11.)
Th" combined apprT.Jac:h to ccntrollin,. malaria has _included tre developnent
of
vaccines~
genetically engineered
IlDSqui t.DE":;!f,
pha.r-lliacB...ltica.ls~
This approach, ro,..,-
il15f'ccticides and physical traps -fO<'- catching fl'lCJSqL.i t,,)(,-'S.
ev.,,--,
and t.tlE? use of
has failed to significantly impr-ove tre qlob.;,l c;ituat_icn (.11.)
M:.-,squi_-
tn::"5 ar-e develDping resistance to Che?Ol.icals which v-..er-e once uf5ed "for their-
centrol.
Ther-e is no malaria vaccine as of yet, b<C'Caus<' tre par-asite has st-oaM-l
a sur-pr-ising irT'llLtnolo;}ical vi::\riability, and vacc.ine str"dtegies which seemed
to gEnetically engineer- lJX:Y;quitoes that do not tr-ansmit malar-ia to r-eplac., tt-.?
mE'S
th<,t do,
w~li_ch
"':>-.lld be a difficult task.
A fDr-eign qene has
tt."efl
inser-ted into fl'lCJSquitoes, but to find tre r-ight W<?apon and p-,t it intD tre
r-ight plac., in tre genCl(fO" to have it e){pr-essed, is scxnethinq for- tb., futw-e
(4. )
Tre p,-oblem wi th malar-ia wi 11 get wor-se in year-s aread and -funding
agencies for"
r~..;ean::h
ar"e I'r"ethinking thE-iF" involvement"
in IllC\l.aria rE'>Searc:h
(11. )
Malar-ia is tr-ansmi Ued via He bite of He
A-1e'!Qhce~_IE'2
<OC.l'_3quito.
[,.,en---
tt-olling tl--.= fnopt-.=les is of gr-eat impor-tance in limiting malar-ia and cc_uld
lead to cCTltr-ol of Dt:rer- .,..::>squi tn spec:if"S t.hat carry infect.ious diseaS£>'3.
and fiJiar-iasis ar-e alsc) """,,,,!uit.o--t.XJFne diS<?as.,os (15.)
[,,o1b-ol of both these
mosqui toes is impor-tant to prevent tr-dnSlnissicn of tre diseases.
Tre Ae£,i,§.
~.J;,ti, will be uc;ed for· thi" study t::ecause it has t:.e.", "",,11 studied and
A.,.
~J. larva€> ar-., par-ticular-l y susceptible to the ~2.. t.,t ._ tOldn.
B.
Bacillu§ .tl"lLw·iQ9.iensi§ ?l...lQ?_p-,- israelensis !j)f1cl Qi#.!lob§l!;t,~cta
I;~~!Jlus tt~dL.J:D!;t:!'f:£!J..2.i~ s ..t!:Jsp. t~ which is a soil tx=tctel'-ium, pn:~dLtcE.~.3 an
insecticicial to>:if"l thi::l.t is sp:3::i f iCc':\ll y leth.a.l to thi? lCi.r'''vEle of Hx::~qui t.OE~~3 i!:"lnd
black'fly~ and which cCflt.ains no h:::'trolytic activit'}-'.
rhi~~, (Tk:":\kr.~:) it thf.?
flD.5t
pru1ii'3ing candidate for an E.~ffec:tivp and E:-':'f-,v.1.n:x'Hra"It.all'''' sD.fe fro<squi to
larvicide (1.)
Th? fr.3.ctOf"" If.7.'thaJ. to (ocY.::;qui.t:o lar-va('~'!1 tJ-);-:? ck?lta:-'"Endob::lNin~
kilodaltcn (kDa) (.1.)
Tht~ 3.fl kilDl""'31? g"ne ·fr-agment enc:cxhnq tJ-e lC'n m-
13~\kDa ITO:'5Cjl.d"t.ccid2d pr"DtE'in hi::l~';
ficBnt t:o;.:icity to t\=4t~
bE.;:€."r)
C}c".)I"1E·:d intD ~'-~. ~;.f?Ji and I···!BE. :;~hC:~""'!""1 s.;igni--
&""TI,i:TI..t.:h 1~3rvaE~ (.1,::2,9.)
l0.rppl.i.cdtJOI": of r.i~_:t.,_'!..:t.~. 'f(Jr'-
biolcl(Ji,c.r::·\1 control has SE'.:'Ver'cIl dl'-a~·Jba.c:k!~; i.ne ludin~J thE-:, j-'J£.':'ITOJ. vtic ~?f.JkDa prnt£:?in
(1)~ thE.:: Y··E«::Ju1.r···E?rTII?ITt that 1."1::. irA..tst tx-:? ,:'".i.ppl.ic?d fr'(,?c~uE?ntl)/ L")f:?C:aus(-2 .it sinks to thE?
bottDm ,3nd thE:' fc':iCt th":":1t .it ,1':::- !:SE;~'n9.;j,tivE\ to ult.~-·a\.li.Dlet li'~lht (.1.0.)
(~
)XJb::nt.ial V-JdY to C.iTCU(fl\/E.1Tt.: ~.:hr."-.:."s:.e pr-T)b1 €-?fTr.::. ,i~::. to qf.-?rlc·?tici:?i.l 1 Y f.::flginE't-=-?f"" micr''O-'"
or·~lan ..i.sfn:::; li.\/ing .in thf::';' uPI::x:~;\t· .. laYE!t-s cd ~""'2\tet-· \l'kt:~t"'f~ m::~.ql.\j.tCJ Jar-\/ae 'fE'EX:/
pn.::xju.CE:' tJ·',a:·::,! to;<.i.n.
tEX::dL.\~~E:'
it.
Cy.:;Y)ol::.'ti::!:ctpt"'·.iEt ~-::tr'e a i,JCX--d cDndidat£:? 'fclY"
.i.-:~. di::::.tr··i.tx.lt€:·:'CJ i.n tr-'e upr£~'r lixy'E.;:r··!:;; C::'i:
\t'JE:\tE'I'""
to
this PI'TCE-duy-f.?
(.1.n)
The r::,OkDd gf.:?n~.? has I:*:?t~"'f""l e){r_:w·E..~:;:.~?d .in I;.;._~_ t;;.G?J..:t i::<\nd in tl'-e cY,;:'WlotkicteY".ium
in l~_ ~.Q..Li -i:.ind i"Si~~Jni'fic:ant to.~-:ic.ity to mc)Squ.itcx~:;
\.·JaS
shc(....,~ but the
e:-:pn=.,,==.~:;i.cn of t.D;<i.n in (gf.!.~'f:~Lt~~ q~.\9.f1.t:j..pL~.f:;..~.t.h.!m F'F(-L IrJaS nDt Significant,
the prCJtE~.in coP'/ numl:::er- i-"-k.i5 vE~ry lcA'J~ and (;In-?d·!:.el'- cjeqY-adatim occ:ur-r-ed than was
setTI
.in
f....
"q.l..~
(.1.)
v(octor pTNTV
plasmid LN"l fragments wen" cloned into tt-E hybr'id
is capable af j:r-ansforming rJOtt1 E;:..,.
,~h.ic:h
cyanDbac:tE~r·.iurn,
\":J2.L\~
f,'.,.t.,.J..~
o ..• r studt,·",
III
9YD.~~.;J:r:.:CQCcu§ FU~
7942 (9.)
c:lC.Tlf-.?S c:arTytn{j tl-e 1::::.0kDa t..CJ~·dn gE-7!l'l(-,;?
~gJ..i
FollOAlinq
and tt-E
traI1s·f(Jrm.::~tic::n
1Nf~r-e ~:>E:lcectJ:?d
into E __
by hybridiz,!:.1.tic:n to
o1igCl·-,ucl£."t)t.id., prnt>2S ccnstruc::ted to hybridize to .,ither thee 5' or 3' portion
of the gene,
Twelve of
t~-,..,.-,.,e ,,~..-....JEd
siqn.if icant tm,ici ty to
~~2
f'§:J.l!.ill.
fourth instc\f' larvae; h:::;:"Je'/er, <:.n1y tWel of tt-ese twelve (p-NX10 and p-NX::;) n;r-
tainf2d t.h':'2ir- plasllid insert. as cells v.ere grO\fl for plasmid I:NCi isola1::ic:.n.
vJhei"j cells of tt-ese
t\l-.lO
C lcnes \l'JE're
e){amined under a
phaSf;\ micrOSC'~ol=-'l€:-';'
it.
appeared that en I y r:HVX.10 proouc:ed a small cry,;tall ine tm,in.
1'1::)S;;t. of ttl\? t.o:-cic reccA'Tlbinc\f1t:s
WE'~\re
unstablf:? due tD segr"E:"gaticnal in-'
stabi l i ty in int-Eri tanc., of tt-E rE.'Combinant plasmid.
e;<pF'E?5sic::n of tl")2
t~1 (J"()rE'~
In addi tion tt-E
stable clc)f'")E:'s needed to be greatly incrl?ased for
signi ficant tm(icity to be available.
Tt-E
t_~J:j-';::Q~,
tien is located in tt;., QE\XIl locus of plasmid R1.
sy"tem elf plasmid stabiliza-
This locus mediates efficient
plasmid stabilizat.icn via pnstseqn=gational kill.inq of plaslOid--frE'E' cells (8.)
Th,> lli)r-B locus cent.ain" two g"<1es, t:£i.".,
killing protein, and
to U,.,
rot
~1CJ},
rrf\1'¥1.
and sok FNAs.
rr/iNA IE>acls to
Q§'~Il
locus.
gene proouc:t is a pot"<1t c:.,11-
gene pnJduct is an anti -<sen<>e RNA canple"Entary
Tt-E nEChanism is apparent in tt-E di ff.,rential per·sistenc., of
In nE'Wtonl plasmid-frE'E' c.,11s prolcng£."CJ
synt~is
of tr1€?S€? cells.
tt-e
§flL ..,ros.?
,~h:>se
of
!:J:?t
persistEoc" of
t:£L
pr·ot.,in, ena.,-ing a rapid and <>electiVE> killing
A-1y L",stabl y inheri ted plasmid l:ecO'TES stabi 1 ized by carrying
This systeTi has b?en testted using di ff.,r·"<1t r.,plic:ons, and
plasmids replicating in E._ <;''21i have b?en ,;tabilized (8.)
Ligaticns and transformations we,'e performed to in<>ert tt-E
mrl1 locus into
pTNT\! and p-NX10. Tr<'If1sfonnants wen: obtained thnJ.Jgh grewth on selective
rredia.
in~rtE>d
WE! are new in UE pr-ocess of making sure t.t-E f§lrl'i 1CJCus has be>en
into
t~1e
'lECtor' pTNTV and tt-E tm,ic clcne pl-WX1C>.
MATERIPLS AND I"ETHJDS
Clmes wer'e obtained t.hat had e:<hibited significant rTDSquitocidal activity
'fYTxn a former master's student L_eah Helvering (9.)
They contain part of the
:3.Bkb fragmc-'f1t of U." L''J:l kDa prutein inser-ted into the hybr'id cyanobact.eria/E.
coU vector- p-fNlV (9.)
g'-"fle
The plasmid pTNlV ccntains ,-m c","picillin resistance
as well as a po.-.erful rightward lambda prO'1Pter (14.)
The
~,,'_
coth ,;t.rain >*<8-1022 ccnt.ains the 1;0",-8 t:!JJJspk stabilizaticn locus
in a fJl..ll tiple c 1C:X'ling
rB;.~i.on ~
Thi-::;} plasmid contains a kanamyc:in resist.a.nce
gEne used fo/'- se 1ec: t.ion .
Transfc)t'mat.ic:ns wer'e perforrreci into c01lpet.ent. cells of U." E.,
~.91_,t
strain
DH5 alpha, ,·,hich lacks plasmid l:N'l and antibiot.ic resistanCE' (Bet.hesda Research
Laboratory. )
8. Growt.h of Cells
~-'- ~,g,u.
was grown in Miller"s Luria Broth (Gibeo, Lt.d.)
Fi ft.y mi.c:roliters
of a glycerol stcx:k was added t.o five 101 of Luria broth and alln,'ed to ,.hake at
37C.
Miller's L.B !'<;Jar' (Gibeo, Ltd.) was used for' plat.ing tx"ans'formati,xls and
re-plat.ing clmes,
The concentrations of antibi,ot.ic::s used for sreci fic:: E.
iI')
ug/ml ampicillin, 25 ug/ml chloramphenicol, and
~-e
~oli
cuI tLlr'es were
ug/ml kanamycin.
Pmpi-
ci.llin was dissolved in distilled wat.er at. 25 mg/ml, filt.er-st.er'ilized,
aliquoted, and fl"OZen at '-2(C.
and tt-e rE"'Binder' discarded.
The aliquots wen' thEn defrosted as requir-ed
Chlor"al1lpr.:.nicol was dissolved in absolute ethanol
at ::'4 mg/ml, fil t.er--sterilizo,"d, aliquoted, froz,,,, at -':XC, and used as needed.
Kanamycin was dis.JOlved i.n distilled wat.er at.
aliquoted, frozen at -2(C, and used as needed.
l(x)
mg/ml, fil ter"--st.er-i,lized,
F'12\Ung bacter-ia wer'c' gr-c:JM1 in Lamtda br-oth media
0.201. mal tose.
en,
supplemented with
Lamtda phage "",r-e gr-own in Lamtda tDP aqar and en Lamtda plates
(3) at 37C.
C.
Plasmid I,;olatien
E. ,;p).i pJ.aSlllids •.e"," isolated by a modi i'led metJDd of lar'q"'-'scale ,,<lka'"
line lysis as descr'ibecl by M..miatis (.12.)
A f.i.v>' 101 overniqht cuI tur-e was
added to 3:'.9)-5«> 101 selective media, ampl H.ied with chlor'amphenicDl after' 5-7
ha..tr's of qn::Njth, and
to qr-ow overniqht.
all~
fuq.,.::! fm" 10 lOin at 5-7K rpm.
The
The supernatant was
c~:X}
101 cuI tur-e
~x)l.ln:,"d
cenb-i-
''laS
off in bleach watt"r-
and the pellet fr'o;:en for- tn,,, how', or- lc-..oqer- (fneezinq is opticnal but may
help in lysis of tre celli::'i.)
B..lffle,- (w/v 251. sucr-ose;
pf?llE~t
ThE?
C~:'rM
was
r·E.::-sl.t~;pended
in
Tr'is"+Cl, r:ti 8.0) and j:<Jt 0"
fr-",,';hly made 1 "'J/m1 lysozyme in
2~j(>rM
fj,v€~
ice.
ml Lysing
Ch2 101 o·f
Tr-is+Cl, rH 8.0 was adeled and swidc'C!
gEntly, making sur'e ly=,,(J2ytne was rni,.,,=ci in well.
After- 5 min .. 8 ml of freshly
prepared lytic: mi,·, ( fluJ. Trit.on-X, 2 ml 0.2!."M EDTA, 0.4 ml TF"i.s+Cl, 5.6 ml
f-PLC water) was added.
6<)-·90 min.
It was .incubated cn ice and ,;tilTed C''ccasionall y for-
It was t.hon c:entr-ifuqed for- onE' h:ur- at 171< and 4C.
The superna-'
L'1Jlt was r-BTDved and e,,,tr-acted at least f.Jf1ce with 1/2 vDluff'" phenol and 1/2
volLure c:hlor-ofor-m savino U'<?
vol UJTe chI ot'~ofor-m to
r-E11DVE~
l..Ip~:er·
phase.
This was ed.er-ac:ted t.WiCE' with one
pherno 1 .
The nucleic: acids wer-e ett1af101 precipitated wit.h t.wo volumes
and J.l10 volume
:]"I
sociiL"" aceta·te.
fuqation at. 71< and --4C for' 10 min.
l(X}%
Et.t1af101
The nucleic acids "",r-e obt.ained by cf2ntr'i-The pellet. was washE'<J with 701. et.hanol
twice to rBTDVE' sal ts.
HE pellet "",S saved ,,,,"d tl'E WC'lUS of the tui:".e dr-ied
c",r-efull y with a swo'\b.
ThE> pellet. was
'7.4 101 of lXlE, 8.4 q of CsCl, and
densi ty was adjusted to 1. 57 O.
all~
t.o dry and then br-ought up in
5<X, ul of 10 'TX.]/ml ethidiL"" br-omide.
TtlP
To obtain c1p;;,r bands of c:hrc.:m:JSnlBl and plasmid CN'\, thP sl.lspensicn w,;,S
n"." lC:<-J<?r- plasmid band was
ul tracentr-i fu'lPd for- 17--21 haL..-s at -4C at 42rp-n.
f~-'llpcj
with a syr"in'lp i.lnd dPdyPd usin'l isopropClf)ol/s.'SC.
ThP ,..amplp was dia--
1 yzPd threp tiflles with 1XTE to re<llDVP thP CsCl, with th2 last dialysis 'loin'l
overni'lht.
ThE' S<\Jllple was ther", ccncentrah-;d wi t.h n-"butanol to (>.5 fill, and
et.hanol precipi t.atPd.
ThP CCnG'"1tr-at.i.o."1
waS
detenninPd by a'larose 'leI electro--
phoresis wit.h samples Df kno.", u'l/fIll ccncEntr"at.icn.
Tw:" ,,,,JdifiPd rap:Ld
i1Bt~<:ds
of plasmid
sera,,-i plasmicis frOll transfO,"""nBt.ions.
I:N~
prepar,aticn were employPd to
Appr"O"imatel y 1.oi fill c)f a cuI t.urE' of
[2c"ter"ial cells tr.Ier-e sj;:(Jn dov-.n in a rnicr-ofuge ,"md
~,1.
buffer (8"1. sucn",""",
1yse the cells.
Trit.cn--X 1(X) ,
~'<:"'rM
n,,~·susp=ndt.=-d
in 50 ul of STET
EDTA and ::<:>ir1'1 Tr"is-+Cl, f:H 8.0) to
TtE tut:es were placPd in a boilin'l wat.er bath for '10 seccr1ds
"ttl cienature th? I:N4.
The t.ube was spun at. 12(XX'Ng tor" 10 fIlin. to pellpt d"Tla-
turPd chn::moo3Olnal DIW4, cell M=mbranes and othf2r debris.
The SL'pen1at.ant. was
n"f1DvPd car"efully wit.h a pip.,t, and thP pla<;,mid 1)1\\<\ pr"ecipitat.,,-.cj in an equal
volull"e of isopropanol.
night.
It was frDz"T, at "--"l(C for- .1/2 h::'Jr Dr at --2(C Dver--
CN'\ was pellet.Pd by spinning at L?(XX',",'l in coldrCXJf11 fIlicn)"flll;)e.
The
P.,U.,t. was washPd with 701. ett1dI101 and r-eSL,spE-ildPd in 1XTE.
nE
11-89.
S"-'C:ond rapid nlE't.h:::x::! is a
rl~'"""
pr"otocol and was emplDYf",j st.arting 12-
It. is fDlU"1d in t'PlECC.Jdlar- c::.lo.JjD"9.,c
~
l,,;<t:xJrgj:gr:v
D. F,estricticn EnzYIT." Digest.ion and Agaros€' Gel
~~
n.)
Elpc::t,"Op~D,-esis
Rest.rict.icn Enzymes wer., r:un::h",ser1 "f'nn GeU1E?Sda
form Inh:,:Irnat.icnal Biotec:h·'iDlexjj.e5i ~ Inc.
~J~1.,.1(
r~esea,-ch
Laborat.ories or
DigestJ.cns uSLlall y cc:ntainecl 1 Ut.;;.I of
b...tf·fer p~··"Ov.ided with EflZyrres~ ~'\I'ld 1-2 units uf Ef1zyrre ~-;er- Ltg
final volume Df
Agan:,sf? 'leI
l.~"i
ul.
crt
~
in a
The tL.,beo; were incuoo"tPd at. :S7C few two haL,,"S.
elect.rop~,,-es.i""
of tr-ansfonnant IlN'l and CN'\ fra'llllE'nt.s Wfer-e
performed in 0.7'1. agarvse gels in 1XE buffer ("o6/rM Tris-basp, :XJni'1 50di.l.un phos--"
phate dibas,ie, and lrrM EDTA.)
Gel electrophoresis apparatuse<5 were obtained
fran Beth.';'Sda F~eseard-. Laborat.ories.
Lan;1E,r gel,; (3':; ,< 1.l.~J em) were r1.U'l at 10
vol t.,; Dven-.ight. and at c~) vol ts the ne><t. ",corning until tt-e bnJ'r<.'pt..."nDl blue dye
",a,C',
at thE' bottc:xn of U ..? gel.
f[)t- 1---2 tx.::ur-s.
5o,,<11m- gels; (21. ,., 8.6 em) "ler'e r1.U'l at 50 vDl ts
Ethidium brDl1lide was added to tho gel pr-ior- to electr-ophon?Sis
crt a cu',c::c"f'.tration of 1 ug/ml.
E.
Ti tering and Plating Df I_amtxla Phage L_ibrar-y
F'lating bacteria, IE~:r,9:,', <-.ere gn:.....-' supplemente;'CI with O.:Xtl. mal tCl'''E'.
r..ells weel"e pellet,,-,j and resuspended in 0.0.11'1 Mc]~304'
in 3M media to di ffeerent eoneentr-at.iens.
L.amtxla phagee were d,ilutE<j
fJ'f th=--se dilutiulS, 1(') ul WOl,S added
to l(x) ul of plating bacter'ia and incubated at 3'1C for- :OX, min.
!:he hLlndn:...;:!
microliters of the SLISfJE<lSic.n was ,.,dded to melted <;elective top agar and pcx..red
mto selective li:vnbda agar' plates Ylhich <-.ere incubate;'CI upside dew-I overniqht. cit
37C.
Plaques that appeared en plates wer-e CC<_U'lted and this numbe,- taken times
Ue dilution factor to f,i.nd t.he titE:'r D'f the librar-y.
F.
Labelling of Dl.igUlucleotides;
01 igonuc leotides; Yle ...'&' pr'eviOlJsl y CU'ISt.r1.ICt.ed by mast.er" s st.udent. L.eah
f'e!ver-ing.
TtE'5e Dligonucleotides are seqUEnces LU'l.ique tD the 5' and ;:(' ends
of t.he m,.cleoti.de SE>quence of the Ie") kDa polypeptide (9.)
Tt-.o'y ''''''re labiO'lled
using t.h" for'wa,-d reacticn of T4 polynucleotide kina<;e to transf'2r tt-e term,ina!
9«rrma-labeled phosphate of ATP to the 5' hydro)·,y 'droLlP
(3.)
The reaction mi,.,ture contained O.OC-M Tris pH 7.5,
of t.he oliqonLlcl">Cltide
O.OlM MgClb O.(x)::M
":!;'7
DDT, 50 ug/m1 [-GA, .1-~(l pmole 5' ends, 50 prole p'--, and :;:0 LU'lits kina<;e.
W<lS
This
incubatE-,j fOt- an h::ur at 37C, and the reac:tic::n was stopped by acldi ticrl of
O.:"M EDTA.
Lhincor'por-ated ATF,:'>2 was separ'at.ed by rHln,1.nq t.he mi){h,.r'e oVer'
Sephade,., c~) c:olLullns Ylh,ich wer-e 'first equil.ibr·ated with .1XlF~.
Five elut.ed
samples were collected in eppendorf t.ubes with five washes of 2<:() ul of lXTE.
Each Se"\lT)ple was cc'-'nted using a E\S'c:kman L8 ::'80/Liquid Scint.illaticx1 cCX_lf1ter- to
ejptermine t.h2 ef'ficiency O"f reaction and riLUnbet'" of COlJnt.s pr"esent..
aliquots with thE.' greatest C:OLU"lts j:E'r~ minute W(:?n= pc)::)l€...;G
used in a single hybridizat.ion.
,").fld
2
}!
Th:::rse
to-] q:m was
Tt.." e,.,tra labE'led oligonucleot.ide
"""5
st.oreel
at-2<:C.
G. Hybridization of Lambda Phage Libr-ary
A laml::xja phagE~ l.ibrary t.hcught. to Ccx-ltdin segment.s of th? 3.8 kb gEne
encoding thE'
r:o
at -3:?C overni'.lht.
kDa proh,in was plated onto lamb:la a'.lar plates, anel incubat.ed
NitrcCf211ulose eliscs Wf2r-e plaCed on top of tt-e plates for-
five min. and mar-ked for oriEntat.ion ,,,i th a needle.
Filters were placed c:olC:<1Y
side up in denaturing solutio-1 (.1.::'(1 NaCl, O.::'M NaCH) -for 5 min., and in
t...:> min.
Fil ters were blotted en p,3.per and allc:w=cj to air dry for :30 min.
Fil ters wer-e placed in an EXC vacuum Dvm for- two hc:x-lrs and then placed in a
"seal-a-m.,al" bag for- hyb,--idization.
Pr-ehybridizatic:n solution (loX sse, 5X Denhar-elt.'s solut.ion, O.05/. so.iium
pympt-ospila te , 1 (X) ug 1101 OOi 1ed HSDN'i, 0 • 5/. 80s) ( 6 ) was added to fi 1 ters and
incubatE.,(j at. 65C fDr onE' hc:x-w-.
The prehybridization solution was rHIDvE'C.l and
tee hybridization solution (6X sse, IX Dt?f1hardts soluticn, 100 ug/ml yeast
tIN", and 0.051. scxJium pyrophosphate) (6) was added cOTI:aining 2.5 x l(Y7 cpm
(yf
"Z .....,
gasrma p-···--dATP labelled ol.i.gonucleob.des.
temJEratur-e \"lith shaking for 48 I--cur-s.
Th? m_i.;.,t.ure ..laS kept at rcx:xn
Hybridization ,,olution was discarded
and fil tE·rs were washed in AX sse/O.05% scxJium pyroprDSphate warmed to -:ve for
'30 min.
T~-e f i 1 ters IA.ere wash=..'l(j at roan tEmperaturE? for ~,O min. i:.md allo",.lE?d to
completel y dry t:efor-e autoradiography.
i
I
IIi
i
II
Ii
I
The Genius syst.em of labeling was also employed in scr-eening of t.he lambda
phage libr-ar; using t.1...., wh::Jle plasmid 4()2-7:2 as ., pr-oiJe.
TI...., kit. "laS obtained
'fr-om &>ehr-inger- Mannheim and t.he pr-ocedur-e was follCJWed as wr-it.t.en.
H.
Autor-adiogr-aphy
Put.Dr-adi.ogr-c\phy was used t.o detect r-adioacti vel y labelled
[)I\i(\.
The dr-ied
nitr"ocellulose fil te,-s wer-e taped to a sheet of paper-, cover-ed with anothersheet. of paper-, and placed ne,.,t. t.o a sheet of film <t<odak diagnost.ic film
X/¥l5.)
To
incn~ase
the intensity of the signal emi tt,,"«j by garrina par-ticles to
the film, intensity scr'eens wer-e used.
It.,." film
'~s
e>:posed at. --70C for' an
.~~,.,
amount. of t.ilTe det.e,·mined by t.he fr-eshlesS of p,C'4.
Film was developed by sub-
mer-ging it for two min in Kodak GBX X'-ray developer-, in Kodak GBX r-apid fi>{erfor t\.o\lO min., and in tNater for- Ole min.
I.
L.igations and Transfor-mations
Digestions wer-e per-formed using t1;,lVtII with pTNTV, peNX10, and i=*<G1C>Z2.
Digests wer-e e>:tr-acted with pt">enol :chlor-ofor-m at .1: 1, and with chlor-oform at
1: 1.
The upper' phase
701. et.hanol.
WC\S
U>c'!1 et.t1C\f1()l pr'ecipi t"t.ed, pellet.ed and washed wi t.h
Tt.,." pellet was brought. up in lXTE.
Digested i=*<OlOZ2 was treated
wit.h CII'F' (c"lf Int.estinal Alkalim? Phosphat"se) pdor t.o lig"hon t.o vect.or-.
Tre r-eact.iC".ns cCfls.ist.ed O'f 5 ug
0.01 unit.
fJ'f
[)I\i(\,
2(~rM
Tris j:J"1 8.0, .1rrM M;JCI2' lirM ZnC1b
CII'F'; ,7I(1d it \>laS inc:ubat.ed at 37C for" ::"() minutes.
Afh.r tt..-,
r-eac:ticn a phenol :c:hlor-ofor-m e>:tr-acticn and chlor-oform e,·,tr·acticn
fOlcmed.
WC\S
per'-
The ,"eaction was ethanol pr-ecipi tated, pelleted and r-esusp"?nded .in
lXTE.
The vector-: inser·t r-atio for- the 1 ig"tion \>laS 2: 1.
45C befor-e the r-eacticn.
The IJNL) wa"· heat.?d to
Tt.,." 25 ul r-eac:tion mi:{tur-e ccntained vector- and
inse,"·t 0'¥'1, ligaticn t .... Her·, t.wo units of T4
[)I\i(\
ligase and 501'1 All" (13.)
r-eaction wc':\s cooled to (C and carried out at 1:C for tl,.o.lO h:Llr"s.
The
DH5 alpha cPlls "",r-p grcw--, in five ml of L.uria ovprnight and used to
inoculate l'X> ml of LL.,-ia th., np;.,t day.
3.~)-6
rD..tF's,
S~""Ul
do,..,n i:i.t
?..(X):)
1/~)
to gn:JW for
\/DILtITX:~ O.~1M CaCl~~?
volLme (2 ml) and left 0.1 ice up to 24 h:x.,rs.
subjected to heat shock at
L.l.wia.
r-pn, r'esL.tspended in 1/2
all~
Cells \AJe1'"e p211eted at :!.O(x) rpn -for- five
let sit en ice "for" ten min.
n?'"_uspEnded in
This cuI ture was
4:r
i:nd
min.
!I
C::e'lls "",r-e
-foy" t",) min. befor-e the addition of .1. ml of
Aft.er- cne h:x.,r- of incubat.icn at. 37C, l<)-:;C,(>O uJ. of cells "",re plat.ed in
select.ive top agar- en sel€.'Ct.ive plates.
J.
Mosqui to Assays
Eo coli clones o.er-e gn:,."n shaking for- si>< h:x.,rs at 37C.
ampli-fied wit.h chlor-amphenicol and incubated ovenlight.
E.
using a ilemacytareter" and diluted L.ur-ia to obtain a 1 )-, lOB
Each cuI t.ur-e was
~gl"!.
"",r"e counted
COL~1t..
Mosqui to
lar-vap "",re assayed at t.he four-th lar-val instar with five larvae in each cup
assayed.
Concentr-ations fr-cxn 1 )-, 10
and co.1tr-ol s "",r-e assayed.
5
up to 1
X
lOB cPlls/ml of tr-ansfor-mants
(he ml of cuI ture or di luted cuI ture was added to
t<:..., ml distilled water "u1d 0.1. ml of 101. liver pcw:Ipr- solut.ion (1\\._'tr1 t.i.o.-,al
Biochemical eor-poratia1) in 1 oz. snap cups.
24, 48, and 72
K.
L.arvae mar-t.ali ty was sconxJ at.
h..-:x..IF"S.
Page Gel Elect.rophoresis
A gr-adient.
~~)l yacrylamide
gel was
rL~
t.o det.erminp sizes of di ff'~n'nt.
proteins produced by clones showinC) t.o)dci t.y.
A C)radient gel was pJured
ccnsist.ing of a .12'1. gel (15.:; ml 201. acrylamidp, 3 m1 deHnized wat.er, 6.5 ml
1.:=M Tris pH B.B, 125 u1 of 201. 80s, 20 u1 of TEl"ED, 125 u1 freshly made
O.lmg/m1 ammc:niLun persul fat.e) pc• .tred into the ex_ ,ter- chamber cd the gradient gpl
apparatus and .101. gpJ.
(1~~.:5
,"1 2(>1. acry1amid,;>, /, m1 de1cnized water_, 6.25 ml
1.:=M Tris r.H B.B, 125 uJ. 201. SDS, :'X> ul TEM."D,
1.2~_i
ul O.Jmg/ml a/TYIlalil.un per-
r
..,.
sulfate) fJC>-Ired into U". inner- chamber.
ThO? gradient gel was fJC>-Ired into tt-e
plates of a ft:effer' Scienti'fic InstrUffB1ts apparutus allowing E<1ouqh roc'" for
tt-e stad:,ing qel.
HE gel was allcw?d to solidify for haH an ho.J'- with water
on the top to ensure an eva"j gEt}.
Tris, 5101 distilled "!ater-,
~O
Stacker gel
(2.~1
ml
acrylamide~
2.5 ml
OR~t1
ul 201. SDS, 6 ul TF.:l'ED, and 50 ul allm:nium per-
su.lfate) was preparED am:1 added aft.er rE'fOClval of water' to top of resolving gel.
This was allcw-,d to <3C)l.idi fy for :,(>--<'K) min.
,o;nall F'AGE qels wer'" also made which consisted of a 101. resolving and
stacking gel.
H-ese ",.ore made according to the procedure fatnd in !1;J.lecul<;\!:
F'r-ot.ein samples wen,< pr-.'pareel by micn:x::entrifugaticn of 1.5 ml of Luria
cuI ture 'for 1 min. and resuspension of the pellet in l(x) ul o'f sample J:x..lffer.
Samples and rrolec:ular' """ight markers were boiled for 5 min and loaded 01to tt-e
gel.
'"i
'.
I
TI-:e remainder- of tt-e sample was frozen at. -:2<:C.
FiESlLTS I'ND orSUESICN
A.
lsolatien and Analysis of Plasmid I:lN"l Frcxn Tm·,ic Clcnes
To cenfirm tt-e presence of a
12,_1....;&.,.
·fr·agrfEnt in tm<.ic clenes, plasmid I:lN"l
was isolated and subj,,,cted to restr'icUen digestien with !;..r:;.QRI, !i.j,UgI II, and
XI:@I.
A:]arose gel electroptDresis ,;r.c....,-:d that the clenes p-NXI0 and p-lVE44
centained inserts into tt-e vector pThfTV as of ~>-18-·B9 (Figur-<"e
1.)
A gradient
r-01yacrylamide gel also shr::w;::d that tt-e clene pl"NXI0 was producing a protein
different fran HE vector pTNTV.
ThrDuqhout HE SLtm11E'r, tl-oE, plasmid I:lN"l from
tm,ic clales wa,; isolated and sr.:..,n to not centain a plasmid insert, implying
B.
Screening of Lambda Phage Library
A Lambda phage library censtruc:ted to cent.ain pat-ts of tl-oE" 4Q2·-72 plasmid
ccntaining tt-e l:::.r'()kDa toxic pr-otein
fragrrent. crf t.t-e £<... t:.,-~., endot.m,in.
tN<3S
used as a r-eservoir to obta.in a to>:ic
Radioac:ti vel y labeled oligenuc leot.ides
sf:.eci Hc: to HE 3' and 5' f=lld,; of t.t-e .1C'(lkDa gene were used to probe t.he
library.
8i,., plaques were obt.ained fn:m HE initial hybridizat.ien screening.
Secendary scn:-€f1ing, rOol8ver, did not ,;Ilc::w up any plaques which hybridized to
th: probes.
HE Genius syst.em was uUlized to latEl t.he 4CQ-72 plasmid to determine if
the plaques ccnt.ained any of tt-e plasmid i t581 f.
In this hybddizaUa1 HlB out.
206 plaques hybr'idized t.o t.h;, anUgen labeled 4C12--72 plasmid (Figure ~':.)
FLtr"tl'-er' analysis of tt-e library she....ed that tt-ere ...ere not to)-(ic fragments
emtained in HE library.
Fur·HEr r"..,al ysi,; of Plasm.id I:lN"l of Tm,ic elenE'S
To ca1firm tt-e absence or' presence of a fr"agment in pl",smid
r.N'\,
HE
plasmid IN'1 fran tl..... clc,..,,'>S r:HJX:::', p-NXIO, p-M::.24, and p-lVE48 was isolated and
en aganJ"'.E qels with tt-e or-.i.ginal plasmid vf£tor pTNTV.
TlE £,001 clcnes
r
Figure 1
.
-
)
....•.......
-;
~~
Ethidium Bromide staining
patterns following agarose
gel electrophoresis.
The
size of the lambda marker
DNA fragments are indicated
on the left of the gel.
Each lane contains
1 ug of
specified DNA, unrestricted
or restricted with specified
enzymes .
•
kb
23.1
~J
~'-"').
:.-~
--:-
.
~
.~
•
••
•
2
•
""""
phVX10 ;"'I1d phVX::, ran di·Herentl y frc-.m p"fNTV, sl"XJlcJing they are not the vector,
cut do contain an insert.
Tt-e !;:£gU cla·\e5 p-M24 and r:Hv'E4El ran the same
length as the vector pINTV
(f~igur."':;
D.
3 and 4.)
Inserticn of the J;&rB L.ocus into pTNTV and phVXI0
The R§r.:B locus of ",,<G1022 was e,·:cised wi til the restriction enzyn;:" BI'M-II.
The vEct.or pTNTV and clone p-NX10 were cut wi t.1l BantU.
L..i.gaticr.s with thE!
vector and b§r:.f2 locus inser-t n?sul ted in kanainycin resisti:.V1t transforma.r1ts.
T.JEnty-·five transfor"mants we,·e obta.iIlPd ftTlI1l tl-", ligat.icn with rt-IY'X10 ,o;nd
str-eaked
c..'V1tO
ka.r1a.mycin
n0..;i~5taJ1t LLlr~ia
plab-:::s.
L.uria with kanamycin for fw"·Uer· grDwth.
Tr",~.1nsformants
\!'Jere
~:t.lt
in
The clone r:H"Xl0--14 was shcw1 to 00
able to 'lrow; hovJEve,", it had very slow g,"owth taking apprm:imately five days
to qrDw up. E.
E.
g~.th
Phase Mic,"oscop" Ix,t.ect.i.cr\ o·f Crystals
cell s gn:W-l up overniqht of tt-e str"ains pTNTV, phVX10, and phVX3
were anal yzed under· the phase micF"C)sc:ope to see if a crystal toxin waS
apparent.
The cla1e rHJX10 cells did show a small tm:ic crystal which was
"OC1Ut 1/3 the size of the
~"<
£;oli cell,;.
of the i;;.... £gli cell,;, tut in par"t of tt-em.
phVX3 did not show any tm:ic crystal.
Thi,; crystal wa,; not apparent in all
The cells of the strains pTNTV and
The clcrle phVX1C>--14 waS grov.n for five
days and also looked at under tt-e phase micn:>scope.
crystal whi.ch
""*"'""""
Tt-ese cells did centain a
to be apparent. in almost all of the E •. coli cells.
The
higher· e,qlF"e5sia1 of t.l-e t.m:in may 00 due to the insertion of the PSl!::.;'
sequence.
Further analysis of this clen'" will t::€, m;;ndatory to tell what is
ccntained in the plasmid.
....
Figure 3
"
Ethidium bromide staining
patterns following agarose
gel
electrophoresis.
The
size of the lambda marker
DNA fragments are indicated
on the left of the gel.
Each lane contains
1 ug of
specified DNA .
•
kb
23.1
3.4
6.6
4·4
Figure 4;
Ethidium bromide staining patterns
following agarose gel electrophoresis.
The size of the lambda marker DNA
is not labeled due to the fact that
it was not
run
far
enough
to do so
but is just a quick analysis to
compare the relative sizes of the
plasmids.
Each lane contains
lug
of specified DNA.
~ rtl
Q
r
~
C.
r
•
I 0 /1Y1'4" "s:\.
'
IIi""'\
4
CXN:::U.JSICl\IS
~'n
envir'onmentall y safe way t.o centl'"ol malal'"ia has teen pl'"oposed by tt-e
use o'f !3-!t.i. in cyanobactel'"ia.
Tt-e e,,:pr-essiO'1 ()'f tt1e to)-:in crystal has not
been stable encx.lgh to be signHicantly tm:ic t.o ffOSquitoes.
~.,
"_QJA clenes
have shOl..".., that tr.,-,y al'"e not st,.ble and cyanobactel'"ia cla1e<s do not have high
encx.lgh e'''pl'"ession of tt-e t.o,<in t.o be significant.
Tt-e denes which had beE" st-.:::ov.n to be tONic wel'"e mostly 5egl'"egatienall y
unstable.
p,-ot.f~in.
This may bo, due to
Tt-e
~.ff.f:I
irwel'"t~:d
I'"epeat. sequences al'"ound tt-e lC'OkDa
fl'"agfrent may have centainEd tt1e inv"l'"ted I'"epeat sequences,
when,as the X,,99l1 fr'agfrent might not. have, due to an X,99l1 site in tt-e 3.8kb
'f,-agrflE.>flt.
Tt-e da1es p-NXlO and p-NX::: hyb,-idized to th." 5' and 3' ends I'"espec-
tively of tt-e lC'OkDa gene.
This could 00, a r-easa1able e;:planatien of why the
Xb.j;I clcnes wel'"e st.able wt-e,-eas tt-.e Ecr:t<I clenes vel'"e not.
Tl'"ansfol'"matien o'f t.t-e vector' pTNTV wi th tt-e P.,oI'"11 I a::us was not successful.
TI'","\nsfol'"matiol of tt-e tm:ic cla1e p-NX10 did produce tl'"ansfonnants which gr-ew
on kanamycin r'esistant plates, and (T,e transfonnant p-NX10-14 which gl'"E'W up
sepdl'"ately in kanamycin resistant. Luria.
Tt-e clo'1e, i-<:JWeve,-, has taken five
days t.o gl'"DW up and does not grD."J in ampicillin.
Tt-e Q£\.rB locus does not seem
to be a bettel'" way to stabilize cx.ll'" clcnes, althaJgh it appeal'"ed to be
producing rron? cr-yst.als undel'" tt-e phase microscope.
FUI'"t.t-er analysis of this
clene will be nec:essar-y to I'"eveal what. t.t-e plasmid centains.
FL..-tt-er st.udies in this ar'ea ccx.lld
by p-NX10 and p-NX3.
c:hal'"act.eriz~'
Mosquito assays ccx.lld be
tt-e
perfom"~
poly~,ptides
produced
using p'NX10 and p-NX:3
separ'atel y and togett-er to see which sequence produces t.m:ic pl'"ot.eins Dr if tt-e
pol ypeptides produced by tt1eSf? two
"~Jrk
togett-er in pr-oduc:ing tm:ici ty.
Fur-ther- wor-k ccx.lld also be dene in transfor-ming cyanobact.eria with th", plasmid
DI\I'\ f ,-om tI'-ese two to)-: i.c c 1alE.,; .
r """
LITERA1l.f>E CITED
1.
i'ngsuthanascmbat, C., S. F'anyim. 1989. Biosynthesis of IS0'-kilodal ton
t1::>squito Larvicide in the (,'yanobacter-ium ~!&fl",JJ!dffi glJadtJdl?li>=al;.~'ffi F'R-6.
Bm+-.ieg @[!g
"'
..::.
.
~viCfD~tal
MiC;TObiQlj;gY:. ~~
~~::;:~4:28-··:':~4~~,O.
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