Thomas Wang
October 13, 2002
Mtg. 3, in class exercise
(completed out of class)
Results for Exp. III-B
Scoring Ara and Lac Phenotypes of Transposon mutants
Results:
We patched from colonies from each of our five experimental BW140/pNK strains that
underwent transposon mutagenesis along with controls on five different plates to determine ara,
kan, and lacZ phenotypes. Our 4 controls gave expected results for the patching on the five plate
types. The control for strain BK1 (ara-, kanr, lacZ+-inducible) gave an important qualitative
result. Though the strain has an arabinose-induced lacZ fusion, there was evidence of B-gal
activity on both the LB Xgal Kan plate and the LB Ara Xgal Kan plate; however we made a
distinction in color intensity. On the plate with no arabinose, the color was faint blue, and on the
plate with arabinose, the color was dark blue. For our experimental results we could expect a
similar result when distinguishing between lacZ+ constitutive and inducible phenotypes.
Constitutive phenotypes should have little or no difference between the blue tone on the two
plates, and inducible phenotypes should have the observed tonal difference as with BK1.
Grid #
1-4
5-8
13-16
17-20
29-32
33
41
47
50
Strain
J1W1
D3W1
B1W1
J3W4
R2
BK1
Jet2
H24
EJ1
M9 Ara
Kan
+
+
-
M9 Glu
Kan
+
inconsistent
+
inconsistent
+
+
+
+
+
Mac Ara
Kan
+/white
+/white
+/white
+/white
+/red
+/white
+/red
+/white
+/white
LB Xgal
Kan
+/ lt. blue
inconsistent
+/ lt. blue
+/blue
+/white
+/ lt. blue
+/white
+/blue
+/white
LB Ara
Xgal Kan
+/ dark blue
inconsistent
+/ dark blue
+/blue
+/white
+/ dark blue
+/white
+/blue
+/white
We plated four ara- mutant strains and one ara+ mutant strain. R2’s phenotype is ara+, kanr,
lacZ-. The R2 strain grew on both minimal media plates (M9 Ara Kan and M9 Glu Kan). This
confirmed insertion of the Kanr and lacZ genes from the miniTn10 transposon in a non-Ara gene
in the recipient BW140/pnK strain. This mutant also appeared red on the Mac Ara Kan plate due
to its metabolism of arabinose sugar, which lowers the pH and leads to the red color. The strain
appeared white on both LB Xgal plates (LB Xgal Kan and LB Ara Xgal Kan), indicating that the
lacZ gene was not inserted in the correct orientation or reading frame or in an open reading
frame. The kan gene is expressed regardless of the specifics of its insertion because it has its
own promoter gene.
We cannot conclude any phenotypic characteristics from our D3W1 and J3W4 strains. Both
strains gave inconsistent results on at least one plate. This indicates that our strains were not
pure since different colonies from a single strain gave different patching results.
The remaining two mutant strains, J1W1 and B1W1, gave the same consistent phenotypic
characteristics on all 5 plates. These two strains are both ara-, kanr, lacZ+-inducible. The strains
had no growth on the M9 Ara Kan plate and growth on M9 Glu Kan plate, which confirmed
kanamycin resistance and miniTn10 insertion. The strains had white colony growth on the Mac
Ara Kan plate due to the ara- phenotype. These strains could not metabolize arabinose and
instead metabolized amino acids, resulting in increased basicity and the white color of the
colonies. Finally, both colonies had patching results on the LB Xgal Kan and LB Ara Xgal Kan
plates identical to that of BK1; colonies appeared light blue on LB X-gal Kan and dark blue on
LB Ara X-gal Kan plate. We concluded that these strains are LacZ+-inducible like the control
BK1. We further know that miniTn10 made its insertion into either the AraA or AraB gene
because these are the two inducible genes of the arabinose operon, and that this insertion
occurred in the correct orientation and reading frame.