IGEM:Paris Bettencourt 2012/Notebooks/MAGE group
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looking for MAGE protocols from past iGEM team
MAGE oligo strand choice
Choosing the strand
MAGE is much more efficient if the lagging strand is targeted with the MAGE oligo. It is thought that the MAGE oligo takes the place of the RNA primer that begins Okazaki fragments on the lagging strand during DNA replication. The diagram below shows which strand to choose when designing MAGE oligos. Use ecocyc.org to determine where on the genome your site of interest lies, and which strand it's on (determined by forward or reverse orientation in the genome browser). Examples
- For HisB deletion, we used the same strand, and for the rpoZ deletion, we used the reverse complement.
- Grow up cells to mid-log at 30C.
- Induce lambda red machinery by incubating cells at 42C for 15 minutes (for other strains, induction procedure may be different).
- Centrifuge 1-1.5mL of culture at 4C for 1 minute at top speed
- Remove all the media from the pellet and resuspend in 1mL of cold ddH2O before spinning again at 4C for 1 min at top speed.
- Repeat for a second water wash.
- Remove water carefully, taking care not to disturb the cell pellet. Add the MAGE oligo (amount will vary: add enough to have a final concentration of 2.5μM in a total volume of 50μL) and cold ddH2O to bring the volume up to 50μL. Mix and transfer to a cold, 1mm gap cuvette for electroporation.
-Note: MAGE oligo should preferentially be concentrated enough that only a few microliters need to be added. Adding larger amounts, especially if the salt concentration is high, may interfere with electroporation.
- Thoroughly dry the sides of the cuvette and electroporate at 1.80 kV for ideally 5.7 ms (Ec1 setting on many electroporators).
- Immediately add 1mL of plain LB to the cuvette, pipette up and down to mix, and transfer to a culture tube containing an additional 2mL of plain LB.
- Recover at 30C at least 1 hour before plating, or if performing additional rounds of MAGE, let culture grow until it reaches mid-log and repeat procedure.
- "Received 22 degenerate oligonucleotide sequences from Keck and performed MAGE. Cultures were grown up to midlog post electroporation and freeze-thawed approximately 14 times before plating. Suriving colonies were isolated, and we will soon put them through another cycle of MAGE. The process will be repeated. "
List of Auxotrophic Strains
- Alanine There are multiple pathways for alanine biosynthesis, and no known complete auxotrophs exist.
- Arginine Knockout in argA (Strains JW2786-1 and NK5992)
- Asparagine Requires knockouts in both asnA and asnB (strain ER)
- Aspartic acid Requires knockouts in both aspC and tyrB (strain DL39)
- Cysteine Knockout in cysE (Strains JW3582-2 and JM15)
- Glutamic acid Requires knockouts in both gltB and gdhA (strain PA340)
- Glutamine Knockout in glnA (Strains JW3841-1 and M5004)
- Glycine Knockout in glyA (Strains JW2535-1 and AT2457)
- Histidine Knockout in hisB (Strains JW2004-1 and SB3930)
- Isoleucine Knockout in ilvA (Strain JW3745-2 and AB1255)
- Leucine Knockout in leuB (Strain JW5807-2 and CV514)
- Lysine Knockout in lysA (Strain JW2806-1 and KL334)
- Methionine Knockout in metA (Strain JW3973-1 and DL41)
- Phenylalanine Knockout in pheA (Strain JW2580-1 and KA197)
- Proline Knockout in proA (Strain JW0233-2 and NK5525)
- Serine Knockout in serA (Strain JW2880-1 and JC158)
- Threonine Knockout in thrC (Strain JW0003-2 and Gif 41)
- Tryptophan Knockout in trpC (Strain JW1254-2 and CAG18455)
- Tyrosine Knockout in tyrA (Strain JW2581-1 and N3087)
- Valine/Isoleucine/Leucine Knockout in ilvD (Strain JW5605-1 and CAG18431)