(r7) ProtocolsAraMarker < Lab

---+ The Arabinose (Ara) Genetic Marker

---++ 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<sup>&minus;</sup> (REL606-derived) and Ara<sup>+</sup> (REL607-derived) cells in a mixture for competition assays or marker divergence experiments. Ara<sup>&minus;</sup> and Ara<sup>+</sup> 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* | *<i>araA</i> sequence* | 
| REL606 | Ara<sup>&minus;</sup> | 92D (GAC) |
| REL607 | Ara<sup>+</sup> | 92G (GGC) |

Selection for arabinose utilization from REL606 is known to also produce, more rarely, Ara<sup>+</sup> revertants that have an _araA_  92A (GCC) sequence.

---++ Selection of Ara<sup>+</sup> Revertants from Ara<sup>&minus;</sup> Strains Derived from REL606

For a non-mutator strain. Grow several 10 ml cultures in LB or DM1000 media. Spin down to concentrate cells and plate them all on separate minimal arabinose (MA) plates. Grow for 48 hours, pick one colony from each plate (it has to be an Ara<sup>+</sup> revertant to grow). Streak each candidate colony on a new MA plate and grow overnight to verify that it is Ara<sup>+</sup>  and a clone. Then, grow an overnight culture in LB or DM1000 and store frozen.

---++ PCR-RFLP Assay for Verifying the REL607 Mutation

To verify that you have the precise reversion present in REL607 in your new Ara<sup>+</sup> revertant candidate, you can use PCR followed by digestion with a restriction enzyme that cuts only when the REL607 allele is present.

Perform a standard PCR reaction from whole cells with these two primers.

| *Primer* | *Coordinates* | *Sequence* |
| REL256 | REL606/70660-70683 | 5'-CCGATACGCTCATGGGCTTGTTTA-3' |
| REL257 | REL606/71177-71154 | 5'-CTGCCCAGGCCGTTGCGACTCTAT-3' |

Cut with _HaeII_ and run on a 3% agarose gel. Ara<sup>+</sup> with the REL607 reversion mutation will show a shift of the 226 bp band down into the composite ~200 bp band because this mutation creates an additional copy of the restriction site.

| *Strain* | *Marker* | *RFLP Fragment Sizes* | 
| REL606 | Ara<sup>&minus;</sup> | 226, 197, 72 (bp) |
| REL607 | Ara<sup>+</sup> | 207, 197, 72, 19 (bp) |

The expected result is that a majority (>50%) of selected Ara<sup>+</sup> revertants have this exact mutation.
Barrick Lab > ProtocolList > ProtocolsAraMarker
Topic revision: r7 - 2010-09-20 - 23:46:15 - Main.JeffreyBarrick
Lab.ProtocolsAraMarker moved from Lab.ProceduresSelectingArabinoseMarkerRevertants on 2010-06-10 - 18:13 by Main.JeffreyBarrick -