Biology Reference
In-Depth Information
Make up to 50 mL in dH 2 O, pH to 7.0 with KOH and filter sterilise
1. Inoculate 10 mL of MD medium supplemented with 50
L of 20% (w/v) of
casamino acids with B . subtilis to an OD 600 of 0.2 from a freshly struck plate and
incubate at 37 C with shaking until OD 600 of 1-1.5 is reached.
2. Add an equal volume of MD medium and continue shaking for a further 1 h at
37 C.
3. Add 1 ng of purified plasmid DNA to 800
m
m
L of the culture and shake for 20 min
at 37 C.
4. Add 25
L of 20% (w/v) casamino acids and shake for a further 1-1.5 h at 37 C
and then plate onto nutrient agar supplemented with appropriate antibiotic.
m
2.1.4 Screening transformants
Screening can typically be done by checking the fluorescence of the transformed
cells and also by the following:
a. Check the C-terminal single-crossover constructs by PCR, using a forward primer
that anneals upstream of the forward primer used in the initial PCR amplification
of the gene and the reverse primer used in the screening process (anneals to the
plasmid backbone). An example would be to use a forward primer that anneals to
the promoter region of the target gene and a reverse primer that anneals to the
gene encoding the fluorescent protein.
b. In the case of double-crossover constructs into the amyE locus, screen for the
hydrolysis of starch by patching colonies onto a nutrient agar plate containing 1%
starch and incubating overnight. Patch a positive control in a defined location
(amylase positive). In the morning, place a few iodine crystals on the inverted lid
and allow them to vaporise onto the surface of the agar for
30 s. Double-
crossover clones will have inactivated amyE , do not hydrolyse starch and
therefore do not have a halo around the colonies.
2.2 Escherichia coli
Another organism that has been widely studied using fluorescent proteins is E . coli .
As with B . subtilis , the ideal situation is to create a functional fluorescent fusion
protein expressed from the natural endogenous promoter. However, a plethora of
non-integrative plasmids exists such as pBAD and pBluescript that can be modified
to contain a fluorescent protein gene that is suitable for the generation of fusions
driven by any number of inducible promoters. These systems are analogous to the
double-crossover integrative plasmids mentioned above for B . subtilis , in that gene
expression is controlled from an inducible promoter, and the intact wild-type copy is
available for complementation of any partially functional fusions. The majority of
common plasmid cloning vectors are maintained at a high copy number in E . coli .
This can make it more difficult to control expression levels precisely. Therefore, the
choice of promoter used to drive expression of the fusion gene, as well as plasmid
copy number, should be taken into account when considering an appropriate level of
fluorescent protein expression.
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