Selecting Arabinose Marker Revertants
Background
The
Escherichia coli B strain REL606 has a mutation in the
araA gene that renders it unable to utilize the sugar L-arabinose. Strain REL607 is a spontaneous revertant of REL606 containing a single point mutation that restores the ability to metabolize L-arabinose. This marker is selectively neutral in a variety of conditions and can be used to determine the relative frequencies of Ara- (REL606-derived) and Ara+ (REL607-derived) cells in a mixture for competition assays or marker divergence experiments. Ara- and Ara+ cells form red and white colonies, respectively, on tetrazolium arabinose (TA) plates, because utilization of the sugar rather than only the tryptone and yeast extract components of this medium causes the excretion of acetic acid which acidifies the area surrounding the colony, changing the tetrazolium indicator color from red to white.
strain |
marker |
araA sequence |
REL606 |
Ara- |
92D (GAC) |
REL607 |
Ara+ |
92G (GGC) |
Selections for arabinose utilization from REL606 is known to also produce Ara+ revertants that have an
araA 92A (GCC) sequence.
Selection of Ara+ Revertants from Ara- Strains Derived from REL606
RFLP Assay for Verifying the REL607 Mutation
Primers
REL256 |
REL606/70660-70683 |
5'-CCGATACGCTCATGGGCTTGTTTA |
REL257 |
REL606/71177-71154 |
5'-CTGCCCAGGCCGTTGCGACTCTAT |
Strain |
Marker |
RFLP Fragment Sizes |
REL606 |
Ara- |
226, 197, 72 (bp) |
REL607 |
Ara+ |
207, 197, 72, 19 (bp) |
Topic revision: r2 - 2008-05-29 - 16:29:28 - Main.JeffreyBarrick