Davidson:Biology Questions

Well it seems we can stop CRE from inverting if we use two mutant LoxP sites. After they invert a wild type loxP site and a double mutant loxP site will be formed which is no longer recognizable by CRE. See Details
 * If HU and Fis are produced at high levels in E. coli, do we need high levels of Hin in order to find the Hix sites? 
 * Which promoter should we use? If we use Lac, then we can turn on for a long time and it will stay on. If we use maltose, then induction will reduce over time.
 * Cre does not need any accessory proteins, then it may be a more efficient system to use?
 * ERIN How can we turn Hin off quickly (using CRE or mutating HIX so that they stop after one reversal)? Off answer
 * B-RAD Do we want to be able to turn Hin on and off more than once?AnswerS
 * Should we create a transgenic E. coli with Hin and/or Cre in the chromosome so we won't need so many plasmids?
 * SABRIYA Does CRE flip once and is then done with that pancake, or will it be excised the next time?
 * ERIC How many flips would the normal negative supercoiling of a plasmid in E. coli allow? What happens to supercoiling if we make the plasmid larger? What happens to supercoiling during the stationary phase, relax? Supercoiling
 * B-RAD Can we alter the amount of negative supercoiling and thus the number of flips if necessary?AnswerA
 * T-ODD Can we apply EtBr to relax the number of supercoils and thus stop recombination?Answer T1
 * ADAM What should we use as the reporters? Fluorescent vs. Resistant pancake or combinations? Only a few points, still unanswered if others want to add more indepth research
 * Possible detection delays for both methods and how to minimize the delays
 * How can we gradually scale up the number of flips with the fewest number of constructs?
 * ADAM Can we use mutated lox or hix sites that will allow only single flips?Answer411
 * T-ODD Will a segment flip multiple times or will the enzymes move to new sites? Answer T2
 * ADAM Which version of hin are we going to use?Answer412
 * Are there any antibiotic genes with biobrick ends in the registry?