Biology Reference
In-Depth Information
1
2
3
4
5
6
7
8
9
10
11
12
A
B
Media
Blank
Media
Blank
Media
Blank
Media
Blank
Media
Blank
Media
Blank
background
strain
background
strain
background
strain
S 1.1
S 1.2
S 1.3
S 11.1
S 11.2
S 11.3
S 2.1
S 3.1
S 4.1
S 5.1
S 6.1
S 7.1
S 8.1
S 8.2
S 8.3
S 18.1
S 18.2
S 18.3
S 9.1
C
S 2.2
S 3.2
S 4.2
S 5.2
S 6.2
S 7.2
S 9.2
D
S 2.3
S 3.3
S 4.3
S 5.3
S 6.3
S 7.3
S 9.3
background
strain
E
S 10.1
S 12.1
S 13.1
S 14.1
S 15.1
S 16.1
S 17.1
background
strain
background
strain
F
S 10.2
S 12.2
S 13.2
S 14.2
S 15.2
S 16.2
S 17.2
G
S 10.3
S 12.3
S 13.3
S 14.3
S 15.3
S 16.3
S 17.3
H
1
2
3
4
5
6
7
8
9
10
11
12
A
B
Media
Blank
Media
Blank
Media
Blank
Media
Blank
Media
Blank
Media
Blank
C1
E1
C11
E1
C21
E21
C2
E2
C12
E2
C22
E22
C3
E3
C13
E3
C23
E23
C4
E4
C14
E4
C24
E24
C5
E5
C15
E5
C25
E25
C6
E6
C16
E6
C26
E26
C7
E7
C17
E7
C27
E27
C8
E8
C18
E8
C28
E28
C9
E9
C19
E9
C29
E29
C10
C
E10
D
C20
E
E10
F
C30
G
E30
H
FIGURE 1.3
Two possible formats for distributing test strains in 96-well plates. The upper format is
designed to examine 18 transcriptional fusions (S1-S18) in triplicate (S1.1, S1.2, S1.3, etc.)
in a single genetic background. The positioning of six control strains (i.e. the strains used to
determine background autofluorescence that do not contain promoter fusions) is shown
(yellow). The lower format is designed to examine the expression of 30 strains in which the
transcriptional fusions are each in a different genetic background (E1-E30), together with a
control strain for each of these backgrounds (C1-C30). All wells should be filled with media,
although the outer wells are not used for culture growth.
integrated at the
amyE
locus to ensure the expression data are obtained from a single
copy of the promoter fusion.
7.5
Growth and data collection
The following procedure was found to give very reproducible growth and expression
profiles. Fresh colonies of strains from the desired storage plate(s) are prepared by
transferring an inoculum onto agar in square plates using a 96-well Boekel replicator
and grown overnight (
Figure 1.2
). The resultant agar plate has a grid of well-
separated freshly grown colonies of each strain (
Figure 1.2
). An overnight broth
culture of each strain is generated by inoculating a 96-well plate (0.5 ml well capac-
ity) with material from each of the colonies on the agar plate using a multichannel
pipette (
Figure 1.2
). Disposable tips on a multichannel pipette are touched to the
