Qs
Rev, 1/55
INSTRUCTIONS FOR CROSSING BIOCHEMICAL MUTANTS OF ESCHERICHIA COLI Kel2

Cultures We6 and Well77 will illustrate the procedure, We6 is auxotrophic for
methionine. Wel177 was derived in several steps, also from Kel2, and carries the fole
lowing "markers": Te Le By= (threonine-s leuciness thiaminerequiring); Lace Mal- Mtl-
Xyle Ara- (non-fermenter of lactose; maltose; mannitols xylose; l-arabinoses--VjP'3ST
(resistant to phage Tl & TS; resistant to streptomycin) « Summarizing the markers,
the cross can be symbolized as:

M TL By Lae Mal Mtl Xyl Ara Vz 5
We wo tet + + + + +88 (F+)
x
Wel177 + ewe - o o - oe FS F (Fe)

The first ), (mtritional) markers are used to select for protetroph recombinants
(M+ T+ Lt By+) by plating a mixture in minimal agar, The remaining 7 markers are free
to recombine in accgrdance with the rules of segregation; if enough colonies are ex-
amined all of the 2/ possible combinations (128) wilt be found.

Sexual. compatibility(S). Until fairly recently, all strains of E, coli K-12
were believed to be mutually compatible. It has now been established that two
“mating types" exist, designated F+ and F-, so that Fe x Fe is sterile, while F* x
Fe and F+ x F+ are fertile, The wild type K-12 strain is F+, as is Wa, while We1177
is Fe, These traits are clonally stable, but mixed cultures of Fe with F+ strain
rapidly result in the conversion of the former to the F+ state.

These compatibility effects can be demonstrated with additional strains that
will be furnished on request. W-2163 is the Fe equivalent of Web; We1817 is the F+
equivalent of Well77, The compatibility can also be demonstrated directly with the
We6 x Well77 cross, by taking advantage of the fact that F+ cultures temporarily
acquire Fe behavior if they have been grown under conditions of 7 eh at aeration
(A), Tks We6 (A) x Well77 will be infertile, while We6 x W-1177 (A) will be fe °

PROCEDURE. Cultures are carried on plain nutrient agar slants, transferred often
enough to maintain viability (3-l; month intervals). Cultures are grown overnight at
37° in any rich broth, preferably buffered. (For example, Difco Penassay Broth),

The cultures should not be aerated or agitated, and should be harvested at the end
of the phase of logarithmic growth or within a few hours, The cultures are washed
in the usual way by sedimenting the cells in the centrifuge, and resuspending in
sterile saline, Two sedimentations will usually suffice. The last pellet should be
resuspended in a smaller volume to achieve a four or five-fold concentration.

After washing, the suspensions of the two cultures are combined, and samples
(.05 = 1 ml) of the mixture are spread on the surface of minimal agar plates (or
poured in deep agar), The plates are then incubated at 37 C.

Prototroph colonies should appear in 2) to 8 hours, against a faint background
of residual parental growth, Although some further tests can be carried out directly
with these prototrophs, for careful work, it is essential, to purify individual
prototroph colonies by conventional streaking methods--the author finds EMBelactose,
etc., the most informative, The fermentation markers are best scored on EMB media
containing the various carbohydrates, V1 is scored by streaking the colonies sus~
pended in 1 ml water across a streak of phage Tl or T5 on agar, 5 is scored simi~
larly by a crossestreaking with a lineestreak of a loopful of streptomycin solution,
1 mg/ml. Obviously, one can score simltaneously for 5 or Vj and fermentation
markers by using EMB or EMS agar.
eo Zea

MEDIA (See also Reference 2)

Minimal Agar (after Davis) Synthetic EMB (EMS)
per liter per liter
Glucose 1 gn. Sodium succinate 5 gms.
KoHPO), 7 Na Cl 1
K HoPO), 2 Ammon Sulfate S
Na, citrate > | Mg 1
FT HD OS eu
. KoHPO), 2
MgSO, ° 7H 0.1
, ; 2 Sugar 10 = 15
NH 1
l a Agar 15
Agar 15
Eosin Y Ook
(Glucose and agar should be
autoclaved separately from salts) Methylene Blue 0.065
REFERENCES

1. Papers in Microbial Genetics. 1951. (selected by J, Lederberg), University
of Wisconsin Press. 21 printing, 1952. pp. 303.

2, Jd, Lo 1950, Isolation and Characterization of Biochemical Mutants of Bacteria,
Meth, in Med, Ras, 3:5=@22,

3. Jo Le an teams E. L, 1946, Gene Recombinetion in Escherichia coli. Nature
158 2558,

he J. L. and Tatum, E. Le 195. Sex in bacteria: genetic studies, 19),5=1952,
A.A.AcS. Symposium, Sex in microorganisms, pp. 12628. (held Dec., 1951).
Also published 1953, Seience 1182169175,

50 J. Les Le Lo Cavalli, and E. M. L. 1952, Sex compatibility in Escherichia coli.
Genetics 37:720<730.

Joshua Lederberg
Professor of Genetics
University of Wisconsin
Madison, Wisconsin