Flip One Pancake

'''Flipping One Pancake'''

The plan is to construct a plasmid that contains the following.  Recombination would be detected as procuction of Tet resistant colonies.
#  Amp resistance gene as in pSB1A3
#  Hin recombinase gene driven by lacP (inducible in JM109 by IPTG or lactose), cloned from PCR product by Davidson team
#  RE (recombination enhancer), synthesized by Davidson team
#  One Pancake test construct:
araBAD promoter, inducible with arabinose
<br> RBS
<br> hixL recombination site, synthesized by Missouri Western team
<br> CDS for Tet resistance in reverse direction
<br> hixR recombination site, synthesized by Missouri Western team
<br> double forward terminator, BBa_B0015 Plate DNA-1, Spot 1I

'''Flipping Procedure (draft)'''

#  Grow JM109 in minimal media to exponential (A600 = 0.5)
#  Induce Hin recombinase with IPTG (how much, how long?)
#  Allow recombination 
#  Turn off recombination spinning down, resuspending w/o IPTG
#  Induce araBAD promoter and Tet resistance with arabinose
#  Spread on Tet plates, look for resistant colonies

'''To Do List'''

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