Flip One Pancake

From 2006.igem.org

Flipping One Pancake

Plan A

The plan is to construct a plasmid that contains the following. Recombination would be detected as production of Tet resistant colonies.

1. Amp resistance gene as in pSB1A3
2. Hin recombinase gene driven by lacP (inducible in JM109 by IPTG or lactose)
3. RE (recombination enhancer)
4. One Pancake test construct:

araBAD promoter, inducible with arabinose
RBS
hixL recombination site
CDS for Tet resistance in reverse direction
hixR recombination site
double forward terminator, BBa_B0015 Plate DNA-1, Spot 1I

Plan B

The plan is to construct a plasmid that contains the following. Recombination would be detected as RFP expression.

1. Amp resistance gene as in pSB1A3
2. Hin recombinase gene driven by lacP (inducible in JM109 by IPTG or lactose), cloned from PCR product by Davidson team
3. RE (recombination enhancer), synthesized by Davidson team
4. One Pancake test construct:

hixL recombination site
araBAD promoter, inducible with arabinose BBa_I13453 Plate DNA-2, Spot 13D
hixR recombination site
RBS, CDS for mRFP, double forward terminator, BBa_I13507 Plate DNA-1, Spot 16N

Flipping Procedure (draft)

1. Grow JM109 in minimal media to exponential (A600 = 0.5?)
2. Induce Hin recombinase with IPTG (how much, how long?)
3. Allow recombination (how long?)
4. Turn off recombination by spinning down, resuspending with no IPTG
5. Induce araBAD promoter and Tet resistance with arabinose (how much, how long?)
6. Spread on Amp plates first, then replica plate on Tet plates, look for resistant colonies

Flipping To Do List

1. Plasmid prep pSB1A3, digest with EcoRI + PstI, Pase, gel purify
2. Anneal hixL, hixR, and hixC oligos, then ligate into vector, transform
3. Start overnights from colonies for double forward terminator, pBAD, mRFP
4. Decide on resistance gene to be flipped, do transformations