Davidson 2006
From 2006.igem.org
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 Department of Biology, [4]
• Laurie J. Heyer Department of Mathematics, [5]
Papers of Interest
• 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.
• The Effects of Symmetrical Recombination Site hixC on Hin Recombinase Function. Heon Man Lim et al.
• The role of the loxP spacer region in P1 site-specific recombination. Ronald H.Hoess et al.
• Non-contact positions impose site selectivity on Cre recombinase. Andreas W. Rufer and Brian Sauer
• Growth Phase-Dependent Variation in Protein Composition of the Escherichia coli Nucleoid
Project Description
Questions to Resolve
General
• What is our team name and project name?
• ERIC What is HU?
- Heat Unstable Nucleoid Protein
- HU stimulates trascription using lambda phage template
- It is made of 2 subunits: HU alpha and HU beta
- It is an abundant non-specific DNA binding protein which is highly conserved between
prokaryotes
• KELLY What is the recombinase enhancer (RE; function, sequence, usual position, relationship with HU, etc.)?
• 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?
- Factor for Inversion Stimulation
- Nucleoid-associated Protein
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?Answer
• 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?
• How many flips would the normal negative supercoiling of a plasmid in E. coli allow?
• Can we alter the amount of negative supercoiling and thus the number of flips if necessary?
• 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?
For in vitro recombination, < 10 mM Mg++ and 30% glycerol traps reaction after DNA cleavage and 2 uM EtBr slows strand exchange. 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
• Where would biobricks be located?
• 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?
• T-ODD Will a segment flip multiple times or will the enzymes move to new sites?
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?
• MALCOLM What is the protocol for assembly for the first stage (restriction digestions, ligations, transformations)?
- [Common molecular reagents]
- [PCR and Mg2+ concentration]
- [Pouring an agarose gel]
- [Calculate MWs]
- [Digest DNA with restriction enzymes]
- [1kb MW markers]
- Shrimp alkaline phosphatase URL to come
- Ligation URL to come
- Transformation URL to come
- Promega miniprep URL to come
Here's what we'd like to see on your wiki page(s):
- A list of all team members, their roles, and email addresses
- Overview of project(s), including schematics and figures
- Ongoing data/updates about project(s), including schematics, figures, test data, and biobrick parts used
- Some photos of your team, facilities, institution, etc.
- Optionally, anything that broadcasts your team's personality, spirit, sense of fun, or coolness...