Davidson 2006

From 2006.igem.org

Revision as of 19:49, 6 June 2006 (view source) Fizzle6821 (Talk | contribs) (→'''Questions''') ← Older edit		Revision as of 19:50, 6 June 2006 (view source) Fizzle6821 (Talk | contribs) (→'''Questions''') Newer edit →
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	[[Math Questions]]		[[Math Questions]]
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	[[Biology Questions]]		[[Biology Questions]]
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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?
-	* <u>ERIN</u> How can we turn Hin off quickly (using CRE or mutating HIX so that they stop after one reversal)?<br> [[Off answer]]
-	* <u>B-RAD</u> Do we want to be able to turn Hin on and off more than once?[[AnswerS]]<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>
-	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]]<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>T-ODD </u> Can we apply EtBr to relax the number of supercoils and thus stop recombination?[[Answer T1]]<br>
-	* <u>ADAM</u> 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]]<br>
-	* Possible detection delays for both methods and how to minimize the delays<br>
-	* How can we gradually scale up the number of flips with the fewest number of constructs?<br>
-	* <u>ADAM</u> Can we use mutated lox or hix sites that will allow only single flips?[[Answer411]]<br>
-	* <u>T-ODD</u> Will a segment flip multiple times or will the enzymes move to new sites? [[Answer T2]]<br>
-	* <u>ADAM</u> Which version of hin are we going to use?[[Answer412]] <br>
-	* Are there any antibiotic genes with biobrick ends in the registry?
	== '''Assembly Issues''' ==		== '''Assembly Issues''' ==

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Revision as of 19:50, 6 June 2006

Malcolm, Laurie, Sabriya, Erin, Lance.

Contents 1 Students 2 Faculty 3 Papers of Interest 4 Project Description 5 Parts Created 6 Questions 7 Assembly Issues 8 Current Course of Action 8.1 Plasmids

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

Parts Created

Davidson Parts

Questions

General Questions

Math Questions

Biology Questions

Assembly Issues

• What is the list of DNA parts that we will need for the first stage, second stage, entire project?
  
• ERIN and SABRIYA Which DNA parts exist in the registry?Answer1More Parts
  
• ERIN and SABRIYAWhich DNA parts will have to be designed? Will they be synthesized or produced by PCR?
  

loxP sites Hin/Hix CRE gene Degradation Tags

• 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. http://www.bio.davidson.edu/courses/Molbio/Protocols/Promegacompcells.pdf Transformation http://www.bio.davidson.edu/courses/Molbio/Protocols/transformation.html Short version
10. Promega miniprep URL to come

Current Course of Action

Plasmids

• pSB1A3
  • • amp resistant
    • copy number:100-300
    • size:2157 empty

• pSB1AC3
  • • part-BBAp1000
    • copy number:100-3000
    • amp and CmR resistant
    • contains device that provides CmR
    • size:3844 ( with device) 3005 (without device)

• pSB1AK3
  • • part BBa_|13535
    • copy number:100-300
    • amp and kan resistance
    • contains a device for screening test
    • size: 3457 (with device) 3189 (without device)

• Transform bacteria We are using Salmonella typhimurium for the Hin protein as it was the referenced species in all of the literature.
• Create RE biobrick (completed)

• Isolating genomic DNA from Salmonella typhimurium
• Ordered olioges for recombinational enhancer and primers for the hin gene