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, neutral 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

---+++ General Procedure

For a non-mutator strain. Grow several 10 ml cultures in DM1000 media (LB is also fine in most situations). 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 DM1000 (or LB( and store frozen. For a mutator strain, you can plate 1/10 to 1/100th as many cells and expect to get the same number of Ara+ revertant candidates.

---+++ Detailed Procedure

---++++ Supplies

| *supply* | *per strain* |
| MA plates | 6 |
| 50 ml Erlenmeyer flasks | 5 |
| 9.9 ml saline dilution tubes | 6 |
| DM1000 | 50 ml |

All quantities are approximate amounts per Ara– strain where Ara+ revertants are desired. They assume three replicates.

---++++ Day 1: Revive

   1 Revive strains to be tested from the freezer in DM1000 in 5 ml cultures in test tubes or 10 ml cultures in 50 ml Erlenmeyer flasks.
   1 Incubate shaking at 120 RPM at 37°C overnight.

---++++ Day 2: Grow replicates

   1 Take each strain you revived (which reaches a density of approximately 5&times;10<sup>9</sup> cells/ml after overnight growth) and make a 10<sup>6</sup>-fold dilution by transferring 10 µl to 10 ml saline in one dilution tube, vortexing, and then transferring 10 µl to 10 ml saline in a second dilution tube.
   1 Vortex again to mix and inoculate 100 µl of this into 10 ml of DM1000 in a 50 ml Erlenmeyer. (You have added approximately 500 cells).  *Do this step 3 times (inoculate three separate flasks) for each of your test strains.*
   1 Incubate flasks shaking at 120 RPM at 37°C overnight.

*Explanation:* Why is it important to inoculate with so few cells? At this stage we are trying to establish evolutionary independence between the different replicate cultures, so that any Ara+ revertants that we find are sure to be derived from independent mutational events, and most importantly, we want to establish that they don't share any deleterious mutations that will prevent them all from being neutral with respect to the Ara– strain we started with.

---++++ Day 3: Plate on Selective Media

   1 Pour each DM1000 culture into a 15 ml Falcon tube. 
   1 Pellet cells by spinning for 15 minutes at 4,000 rpm in the Eppendorf 5810R with the swinging bucket rotor.
   1 Carefully pour the supernatant out of each tube, leaving a few hundred µl of liquid at the bottom.
   1 Using a 1 ml pipetor, pipet up and down to re-suspend the cells in the remaining liquid into a dense milkshake.  
   1 Plate this entire volume on one Minimal Arabinose (MA) plate. You may need to incubate it agar side down if you have a lot of liquid.
   1 Incubate MA plates at 37°C for 36–48 hours.

*Variant:* This procedure is for a strain with a normal wild-type mutation rate. For a mutator strain it is generally sufficient to plate 100–200 µl of a dense overnight culture directly on the MA plates.

---++++ Day 5:

   1 At this point, you should have several colonies on most plates. They might appear as small colonies after only 24 hrs of growth, but it's safer to wait two days before proceeding. Sometimes a plate will have zero cells. Sometimes it might have hundreds or thousands (a jackpot). For more about this distribution, read about [[ProtocolsFluctuationTests][fluctuation tests]].
   1 Pick ONE colony from each plate and streak it out on a new MA plate. This extra step ensures that you have isolated your new strain from the hazy background of Ara– cells and that it is not a mixture of two Ara+ revertants. Store the old plates at 4°C until you are sure your new streaks have grown.
   1 Incubate new MA plates at 37°C for 24–48 hours.

---++++ Day 6:

   1 Pick a single colony from each plate into a test tube filled with 5 ml of DM1000. Store the MA streak plates at 4°C until you are sure your new cultures have grown without contamination.
   1 Incubate cultures shaking at 120 RPM at 37°C overnight.

---++++ Day 7:

   1 [[ProtocolsFreezingStrains][Freeze down each strain.]] Label these cryovials with a temporary strain designation, something like "JEB592 Ara+ #3". Only once a clone passes the PCR-RFLP test to be sure it has the appropriate mutation AND a co-culture competition test for fitness neutrality should the strain be given a permanent designation and [[ProtocolsStrainDatabase][entered in the strain database]].

---++ 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 [[ProtocolsStandardPCR][standard PCR reaction]] from whole cells using these two primers. *Always include REL606 (Ara+) and REL607 (Ara–) controls when doing this assay.* You can use cells from a freezer stock.

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

Use a 1 minute extension time. This PCR produces a 495 bp product.

Next, cut the PCR product with the restriction enzyme _HaeIII_ (Blunt cutter: GG^CC). It is not necessary to use a PCR cleanup kit before this step. To do this, make a master mix consisting of 3.8 µl dH<sub>2</sub>O, 1 µl 10x NEBuffer 4, 0.1 µl 100x BSA (10 mg/ml), and 0.1 µl of _HaeIII_ (20,000 U/ml). Add 5 µl of this master mix to 5 µl of PCR sample. The amount of enzyme per reaction comes out to 2 U. Incubate samples in a water bath or PCR machine block at 37°C for &gt;1 hr.

Run the products on a 3% agarose gel. Load all 10 µl of each sample (adding an appropriate amount of load buffer to them). You will need good resolution of small fragments to see the results. 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: r14 - 2013-06-26 - 02:22:48 - Main.JeffreyBarrick
Lab.ProtocolsAraMarker moved from Lab.ProceduresSelectingArabinoseMarkerRevertants on 2010-06-10 - 18:13 by Main.JeffreyBarrick -