Davidson 2006

From 2006.igem.org

Contents 1 Students 2 Faculty 3 Papers of Interest 4 Project Description 5 General Questions

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]

Project Description

General Questions

• What is our team name and project name?
• ERIC What is HU?Answer1

• KELLY What is the recombinase enhancer (RE; function, sequence, usual position, relationship with HU, etc.)?
• KELLY What sort of spacing can RE allow and still work?
• KELLY Will we need more than one RE to accomplish recombination at all positions?
• TREVOR What is FIS? Answer1a

Math

• 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)

Biology

• 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?
• How can we count the number of Flips? (even vs. odd only?)
• 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?
• B-RAD Can we alter the amount of negative supercoiling and thus the number of flips if necessary?AnswerA
• What happens to supercoiling if we make the plasmid larger?
• What happens to supercoiling during the stationary phase, relax?
• T-ODD (the biologist formerly known as EckDizzle) Can we apply EtBr to relax the number of supercoils and thus stop recombination?

1. For in vitro recombination, < 10 mM Mg++ and 30% glycerol traps reaction after DNA cleavage and 2 uM EtBr slows strand exchange.
2. Doubtful that these conditions could be established in E coli.
   

• What should we use as the reporters? Fluorescent vs. Resistant pancake or combinations?
• 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?
When core sequences are mismatched on the HIN-H107Y mutant, ligation is prevented but synapsis and cleavage is allowed. After recombination, the sites with different core sequences create DNA knots due to "inability to base-pair at crossover site after single exchange of DNA. A second exchange that restores the parental sequence of the recombination sties, but ties a knot in the plasmid DNA, is required for ligation." This would be a possible way to allow for a series of flipping sites to be side by side and keep them from interacting with each other. This question is still unanswered, but interesting info found along the way.
• T-ODD Will a segment flip multiple times or will the enzymes move to new sites?

1. From what we have seen, once an invertasome is formed with two hix sites, recombination can occur multiple times.
2. A new invertasome would have to form with new hix sites to cause a new recombination.
3. Each time recombination occurs, two negative supercoils are used, a topological knot is produced, and the internal halves of two hix sites are exchanged.

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?
• 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

Here's what we'd like to see on your wiki page(s):

1. A list of all team members, their roles, and email addresses
2. Overview of project(s), including schematics and figures
3. Ongoing data/updates about project(s), including schematics, figures, test data, and biobrick parts used
4. Some photos of your team, facilities, institution, etc.
5. Optionally, anything that broadcasts your team's personality, spirit, sense of fun, or coolness...