Davidson 2006

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('''Assembly Issues''')
('''Biology Questions''')
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* Should we create a transgenic E. coli with Hin and/or Cre in the chromosome so we won't need so many plasmids?<br>
* Should we create a transgenic E. coli with Hin and/or Cre in the chromosome so we won't need so many plasmids?<br>
* <u>SABRIYA</u> Does CRE flip once and is then done with that pancake, or will it be excised the next time?<br>
* <u>SABRIYA</u> Does CRE flip once and is then done with that pancake, or will it be excised the next time?<br>
 +
      *  well it seems that we can make CRE
* <u>ERIC</u> 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]]<br>
* <u>ERIC</u> 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]]<br>
* <u>B-RAD</u> Can we alter the amount of negative supercoiling and thus the number of flips if necessary?[[AnswerA]] <br>
* <u>B-RAD</u> Can we alter the amount of negative supercoiling and thus the number of flips if necessary?[[AnswerA]] <br>

Revision as of 19:35, 30 May 2006

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Contents

Students

• Sabriya Rosemond [1] is a rising junior biology major at Hampton University in VA.

• Erin Zwack [2] is a rising junior biology major at Davidson College in NC.

• Lance Harden [3] is a rising sophomore at Davidson College, who might major in math.

Faculty

• A. Malcolm Campbell [http://www.bio.davidson.edu/campbell Department of Biology], [4]

• Laurie J. Heyer [http://www.davidson.edu/math/heyer/ Department of Mathematics], [5]


Papers of Interest

• [http://www.bio.davidson.edu/courses/synthetic/papers/Sanders_04.pdf Stepwise Dissection of the Hin-catalyzed Recombination Reaction from Synapsis to Resolution. Erin R. Sanders and Reid C. Johnson]

• [http://www.molcells.org/home/journal/article_read.asp?volume=16&number=3&startpage=377 The Effects of Ethidium Bromide and Mg++ Ion on Strand Exchange in the Hin-mediated Inversion Reaction. Hee Jung Lee et al.] Excellent description of Hin recombination mechanism.

• [http://www.jbc.org/cgi/reprint/267/16/11183 The Effects of Symmetrical Recombination Site hixC on Hin Recombinase Function. Heon Man Lim et al.]

• [http://www.pubmedcentral.gov/articlerender.fcgi?tool=pubmed&pubmedid=3457367 The role of the loxP spacer region in P1 site-specific recombination. Ronald H.Hoess et al.]

• [http://www.biomedcentral.com/1471-2164/7/73 A high-throughput screen identifying sequence and promiscuity characteristics of the loxP spacer region in Cre-mediated recombination. Perseus I Missirlis et al.]

• [http://nar.oxfordjournals.org/cgi/content/full/30/13/2764 Non-contact positions impose site selectivity on Cre recombinase. Andreas W. Rufer and Brian Sauer]

• [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=103771 Growth Phase-Dependent Variation in Protein Composition of the Escherichia coli Nucleoid]

• [http://www.jbc.org/cgi/content/full/272/29/18434 Hin-mediated Inversion on Positively Supercoiled DNA]

• [http://jb.asm.org/cgi/content/full/185/3/1097 Plasmid DNA Supercoiling and Gyrase Activity in Escherichia coli Wild-Type and rpoS Stationary-Phase Cells]


Project Description



E hop.jpg



General Questions

  • KELLY What sort of spacing can RE allow and still work?
  • Will we need more than one RE to accomplish recombination at all positions?
  • TREVOR What is FIS? Relax before you read this answer

Math Questions

  • What is the problem we are solving?
  • Can we determine more than just Even/Odd number of flips?
  • What is the distribution of the number of flips required, if each flip is random?
  • What designs might allow us to track flips made?
  • Can we model the distance of RE from pancake vs. time or number of flips
  • Can we model the kinetics of n flips?
  • Is it possible to simulate the impact of one-time flipping lox/hix sites?
  • Help us design the fewest number of constructs that will allow us to scale up the number of flips in our constructs (1, 2, 3, 4...n)
  • How can we count the number of Flips? (even vs. odd only?)

Biology Questions

  • 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 that we can make CRE 
  • 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

Assembly Issues

  • What is the list of DNA parts that we will need for the first stage, second stage, entire project?
  • Which DNA parts exist in the registry?Answer1
  • Which DNA parts will have to be designed? Will they be synthesized or produced by PCR?
  • Which plasmids will be used from the registry?
  • Where would biobricks be located?
  • MALCOLM What is the protocol for assembly for the first stage (restriction digestions, ligations, transformations)?
  1. http://www.bio.davidson.edu/courses/Molbio/Protocols/reagents.html Common molecular reagents
  2. [[http://www.bio.davidson.edu/courses/Molbio/Protocols/magnesium.html PCR and Mg2+ concentration]]
  3. http://www.bio.davidson.edu/courses/Molbio/Protocols/pourgel.html Pouring an agarose gel
  4. http://www.bio.davidson.edu/courses/Molbio/Protocols/molwt.html Calculate MWs
  5. http://www.bio.davidson.edu/courses/Molbio/Protocols/digestion.html Digest DNA with restriction enzymes
  6. http://www.bio.davidson.edu/courses/Molbio/Protocols/gels2002/1kbladder.pdf 1kb MW markers
  7. Shrimp alkaline phosphatase URL to come
  8. Ligation URL to come
  9. Transformation URL to come
  10. Promega miniprep URL to come


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  5. Optionally, anything that broadcasts your team's personality, spirit, sense of fun, or coolness...
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