The Exploitation of the Bacillus Subti lis Transforming
System in a Bio-Satellite Experiment
Esther M. Lederberg
May 6, 1963
The ease of handling bacteria, 7’ development of astronomical

populations from relatively small vclum:. of material, and ‘their relative
toughness to comparatively limiting and minimal environments permits their
Inclusion with any other biological materials undergoing - survey since they
do not compete with larger more demanding organisms for space, effort, or
money. Most microbial material intended for experiments of this type allow
certain standardized tests, such as alterations in viability, metabolic
pattern, and the genetic material. The iatter is ascertained by screening
for mutations (preferably reversions) at standard gene loci. The rapidity
of clonal reproduction, and direct expression of novel characters, glve the
bonus that the results of any genetic alteration would become apparent much‘
within a month after retrieval, in contrast to the higher organisms where
several reproductive cycles may be necessary. The serobic spore«former,
Bacillus subtilis is a desirable fest material for stucying the standard
parameters of survival and stabi! ‘ty out!ined above. Moreover at the same
time, an additional and sensitive soratic essay Ts provided by the DNA trans-~
formation system. This system iz = ider «rtloration at the Stanford University
Genetics Department, so the background issoratory machinery Is aval lable.

| in brief summary, DNA is prepa~ eg ine telatively purified form

from donor hacter?® of one genetic constitution and Is then inc:: -d briefly
(3 minutes) with competent bacteria of contrasting genotype wh::' served

as recipients. This ONA thus. represents the fragmented chromosoma: material
extracted Friom the .-ganism in a chssies form, After 24 hours the ensuing
progeny are ready fer classificati.. 0: she segregating markers {genetic
labels). The co-tra"sfer -index of serke : within one DNA molecule. the relative
proportions jand other genetic quan’::ie: .llow certain inferences as to the

size and state of the DNA molecules ‘invcived. * Correlated ‘physico-chemica!

analyses he ve demonstrated that the verlous parts of the chromosomal! materi
can be dif. ‘rent tated and =: arate! by = varlety of measurements. “his u':
mate'y reflects the guanine--yice se cortent of the DNA.
Thus the inclusion of this organism as whole bacteria ana
or, extract provides a unique assay materia! for bio-satellite exploration,
as well as the opportunity for learning more about tye transformation

o
i

system itsel?.
This experiment is intended to test the effccts of the frze-

space environment on the viability and genetic stability of a suitable

Dectcrium, The principal factors of launch and spacecraft environment,

exgcsure to space vacuums, and exposure to solar radiation (penetrating

= Cuartz window) can be treated separately and in combination; comparable

controls will be retained without exposing them to the flight.
Materials:

Controls:

experiment.

These materials can be srepared 2= 4 weeks in advance o* the

YI

imina sketch of reouirements
Preliminary sketch of } t

Frozen competent vegetative cells: multioly-marked mutant.
ca 5 sealed tubes containing 2 ml 107 bacteria.

Lyophilized sample of otA from standard donor (quant)?

Sealed spore samples.

Same materials acked, stored at launch/retrieval cenzer.
» P ,

The volume of material supplied will depend upon estimates

of possible reduction on potency, so that despite this probably inevitable

hazard, enough survivors for the genetic assay will be forthcoming.

Assay of Retrieved Material:

 

1.

3.

Frozen competent ceils: Plate; enumerate; test posuiation
for known and unknown markers.

At the same time transform with standard DNA preparation.
Exemine for change if any in competence; co-transfer index
in linkage group.

DNA

Examine transforming power, co-transfer index, and with
several testers, translocations (unique re-arrangements

of genetic map).

Physical Measurements: Density, melting-point for various
markers, spectra,

Spores: Treat as for 1.
The Laboratory Survey:

The control and experimental packets can be quickly surveyed
and ascertained not long after the samples are returned to the laboratory.
Viability and genetic stability will be known within a week,
and unpredicted genetic changes should they have occurred will begin to

be noticed within that period also.

Instrumental Requirements:

 

The mechanical design and construction of the exposure cans
can be accommodated within the Instrumentation Research Laboratories at
Stanford University Medical School. From 5 to 10 Ibs. of payload weight
would accommodate an extensive experiment. The chief special charac-
teristic is allowance for the opening of scne cans to allow controlled
exposure to free vacuum (harder than obtainable on earth) and the sealing

of the cans against contamination at take-off and landing.

Cost Requirements:

About $15,000 of engineering costs are estimated for prototype
development. The laboratory experiments can be accommodated within existing
funding. |

The responsible experimenter is Dr. Esther M. Lederberg, in
collaboration with other members of the Genetics Department and the Instru-

mentation Research Laboratory at Stanford University.