Difference: ProtocolsAraMarker (4 vs. 5)

Revision 52010-06-10 - JeffreyBarrick

META TOPICPARENT	name="ProtocolList"

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Selecting Arabinose Marker Revertants

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The Arabinose (Ara) Genetic Marker

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Background

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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.

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Selection of Ara+ Revertants from Ara- Strains Derived from REL606

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Selection of Ara+ Revertants from Ara- Strains Derived from REL606

For a non-mutator strain. Grow a 10 ml culture in LB or DM1000 media. Spin down to concentrate cells and plate them all on a minimal arabinose (MA) plate. Grow for 48 hours, pick an Ara+ colony. Streak on an MA plate to verify and purify clone. Grow an overnight culture in LB or DM1000 and freeze down.

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RFLP Assay for Verifying the REL607 Mutation

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RFLP Assay for Verifying the REL607 Mutation

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⁺ with the REL607 reversion mutation will show a shift of the 226 bp band down into the composite ~200 bp band because this mutation recreates a restriction site.

Strain	Marker	RFLP Fragment Sizes
REL606	Ara-	226, 197, 72 (bp)
REL607	Ara+	207, 197, 72, 19 (bp)

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META TOPICMOVED	by="JeffreyBarrick" date="1276193635" from="Lab.ProceduresSelectingArabinoseMarkerRevertants" to="Lab.ProtocolsAraMarker"

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