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- 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)?
- 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?
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
- 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?