Biosafety in the laboratory - VIB

therefore sufficient to check in available catalogues, with suppliers or in strain databases (ATCC,
DSMZ, etc.) whether the JM109 strains belong to these classes of strains. The answer in this case
is: yes, JM109 belongs to the E. coli K12 class.
Next, the vector’s suitability for risk class I should be determined. PUC18 is a well-characterised vector
that has no hazardous properties and is non-self-transmissible. The vector is suited for risk class
I. (a detailed description of the requirements for vectors can be found in annex 2, chapter A to this
booklet. A description of what is meant by “well-characterised” is also given there). It can therefore
be concluded that the combination of this specific host and vector is suited for risk class I.
Step 2: Now the relevant containment category can be looked up in annex 2. In this particular
case, category 1 of the first direction of annex 2, chapter A, is applicable (since the combination
of host and vector is suited for risk class I) However, a final assessment cannot be given before the
risk the insert itself poses has been determined.
Step 3: In this case a piece of bovine genetic material encoding for the production of chymosin, a
cheese-clotting enzyme, is used. It is well-characterised, known to be no toxin, to have nothing to
do with viruses or pathogenic organisms, and to pose no threat in any other way either. It can therefore
be concluded that category j of the second direction of annex 2, chapter A is applicable here,
and that the corresponding containment level is L1.
Step 4: Are there any special details with regard to (1) the environment that might be exposed to
the materials, (2) the type and level of the activities, and (3) any non-standard procedures? In this
particular case only small-scale, standard activities will be performed, such as transformation,
small-scale culturing of bacteria in Erlenmeyer flasks, protein isolation, gel electrophoresis. An activity
is only considered to be large-scale when large volumes are being produced in production facilities.
However, even small fermenters require special attention as far as their leak tightness and the
physical containment of the downstream processing are concerned. In this case no special procedures,
representing an additional risk or requiring a specific form of additional containment, are
performed. The eventual containment level will therefore be standard L1-containment. This
booklet provides the following information on this topic:
• L1-containment requirements: annex 1 to this booklet
• Detailed SMP-procedures: chapter 5 of this booklet
• Decontamination and inactivation procedures: chapter 6 of this booklet.
A possible variation to this example:
Cloning the chymosin gene into a self-transmissible plasmid in E.coli JM109 and transferring
the modified bacteria onto a plastic matrix, during which procedure the formation of aerosols
cannot be avoided.
In this case, the content of step 2 changes: the vector does not meet the risk class I requirements.
As a consequence, the second category of the ‘first direction’ of annex 2, chapter A, now becomes
applicable instead of the first one. The gene product is still well-known and non-hazardous. As a
result, category j of the ‘second direction’ remains applicable and the corresponding containment
level is L2. What is more, a very specific aerosol-producing procedure is being performed, for which
an additional containment measure is required, on top of the standard L2 measures: the aerosolproducing
manipulations should be performed in a class II safety cabinet. The eventual containment
level is therefore L2, supplemented with the use of a class II safety cabinet. This booklet
contains the following information on this topic:
• L2 containment requirements: annex 1 to this booklet
Working with commonly used laboratory organisms 37