Missouri Western State University 2006
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Below are some questions from Jeff and some responses by Todd:<br> | Below are some questions from Jeff and some responses by Todd:<br> | ||
| - | 1. Can we double up on some constructions like Promoter(Forward) and Promoter(Reverse) by inserting them into a plasmid that already has a Hix site and getting a 50-50 split (which we can presumably separate out)? <br> I think it would be better to keep the two promoter orientations as separate constructions. | + | 1. Can we double up on some constructions like Promoter(Forward) and Promoter(Reverse) by inserting them into a plasmid that already has a Hix site and getting a 50-50 split (which we can presumably separate out)? <br> I think it would be better to keep the two promoter orientations as separate constructions.<br> |
| - | 2. Could we start with a plasmid with a Hix site and insert several things at the same time and then sort them out later? (Example: could we put in a short promoter, a medium length RBS, and a long coding region at the same time and sort them out by length as to which connected? I think this is what you referred to as shotgun ligation when we were discussing the Traveling Salesman Problem.) <br> Again, I think that keeping things separate will be easier in the long run. | + | 2. Could we start with a plasmid with a Hix site and insert several things at the same time and then sort them out later? (Example: could we put in a short promoter, a medium length RBS, and a long coding region at the same time and sort them out by length as to which connected? I think this is what you referred to as shotgun ligation when we were discussing the Traveling Salesman Problem.) <br> Again, I think that keeping things separate will be easier in the long run.<br> |
| - | 3. Should Hix be the LAST thing in any subconstruction so that we can create a version with HixL, one with HixR, and one with Hix C? I guess this boils down to the question of how big of a library of parts do we want to create. Is it the case that every time we want to have a Hix site, we need one of each type of Hix? If we take this approach, then we can investigate any differences between the types of Hix sites, perhaps finding the most effective/efficient combination.<br> The way I see it, there will be four pancakes, each in two orientations, and each one will have a hixC on the right (3') end. That way, each could be used in modular fashion to make any of the four pancake constructs we want. | + | 3. Should Hix be the LAST thing in any subconstruction so that we can create a version with HixL, one with HixR, and one with Hix C? I guess this boils down to the question of how big of a library of parts do we want to create. Is it the case that every time we want to have a Hix site, we need one of each type of Hix? If we take this approach, then we can investigate any differences between the types of Hix sites, perhaps finding the most effective/efficient combination.<br> The way I see it, there will be four pancakes, each in two orientations, and each one will have a hixC on the right (3') end. That way, each could be used in modular fashion to make any of the four pancake constructs we want.<br> |
I think that for now we should head for the construction of parts that can be used in the two different one-pancake devices that will work in the four-pancake ones. I think we should concentrate for now on using hixC only. The problem can be subdivided into the following three devices: Hin recombinase producer, pancake flipping operon, and recombination enhancer. | I think that for now we should head for the construction of parts that can be used in the two different one-pancake devices that will work in the four-pancake ones. I think we should concentrate for now on using hixC only. The problem can be subdivided into the following three devices: Hin recombinase producer, pancake flipping operon, and recombination enhancer. | ||
Revision as of 13:08, 20 June 2006
Front Row (left to right) Adam Brown, Trevor Butner, Brad Ogden, Marian Broderick, Todd Eckdahl.
Back Row (left to right) Jeff Poet, Eric Jessen, Kelly Malloy
Contents |
Students
• Marian Broderick [1]
• Adam Brown [2]
• Trevor Butner [3]
• Lane Heard [4]
• Eric Jessen [5]
• Kelly Malloy [6]
• Brad Ogden [7]
Faculty
• Todd Eckdahl [http://staff.missouriwestern.edu/~eckdahl/], Department of Biology, [8]
• Jeff Poet [http://staff.missouriwestern.edu/~poet/], Department of Computer Science, Mathematics, and Physics, [9]
Shipping Address: Todd Eckdahl, Biology Department, Missouri Western State University, 4525 Downs Drive, Saint Joseph, MO, 64507
Research Links
OWW Protocols [http://openwetware.org/wiki/Protocols]
Source Plates [http://2006.igem.org/wiki/index.php?title=Biobrick_delivery&printable=yes]
Goodman et al. 1973-1979. The Pancake Problems [http://www.math.uiuc.edu/~west/openp/pancake.html]
Nanassy and Hughes. 1998. In Vivo Identification of Intermediate Stages of the DNA Inversion Reaction Catlyzed by the Salmonella Hin Recombinase [http://www.genetics.org/cgi/content/abstract/149/4/1649]
Lim et al. 1997. Hin-mediated Inversion on Positively Supercoiled DNA [http://www.jbc.org/cgi/content/full/272/29/18434]
Hughes et al. 1992. Sequence-specific interaction of the Salmonella Hin recombinase in both major and minor grooves of DNA [http://www.pubmedcentral.gov/articlerender.fcgi?tool=pubmed&pubmedid=1628628]
Nanassy and Hughes. 2001. Hin Recombinase Mutants Functionally Disrupted in Interactions with Fis [http://jb.asm.org/cgi/content/full/183/1/28?view=long&pmid=11114897]
Lee et al. 1998. In Vivo Assay of Protein-Protein Interactions in Hin-Mediated DNA Inversion [http://jb.asm.org/cgi/content/abstract/180/22/5954]
Merickel et al. 1998. Communication between Hin recombinase and Fis regulatory subunits during coordinate activation of Hin-catalyzed site-specific DNA inversion [http://www.genesdev.org/cgi/content/abstract/12/17/2803]
Haykinson and Johnson. 1993. DNA looping and the helical repeat in vitro and in vivo: effect of HU protein and enhancer location on Hin invertasome assembly [http://www.pubmedcentral.gov/articlerender.fcgi?tool=pubmed&pubmedid=8508775]
Vasquez et al. 2004. Tangle Analysis of Gin Site-specific Recombination [http://math.berkeley.edu/~mariel/Vazquez_Gin04.pdf]
Merickel and Johnson. 2004. Topological analysis of Hin-catalysed DNA recombination in vivo and in vitro [http://www.blackwell-synergy.com/doi/abs/10.1046/j.1365-2958.2003.03890.x]
R.C. Johnson and M.F. Bruist. 1989. Intermediates in Hin-mediated DNA inversion: a role for Fis and the recombinational enhancer in the strand exchange reaction [http://www.pubmedcentral.gov/picrender.fcgi?tool=pmcentrez&blobtype=pdf&artid=400990]
Lim and Simon. 1992. The Role of Negative Supercoiling in Hin-mediated Site-Specific Recombination[http://www.jbc.org/cgi/content/abstract/267/16/11176]
The Pancake Tossing World Record[http://www.recordholders.org/en/records/pancake.html]
Cool site for Breakfast [http://www.cut-the-knot.org/SimpleGames/Flipper.shtml]
iGEM iDeas
Powerpoint with two possible project ideas. [http://staff.missouriwestern.edu/~eckdahl/Research/Synthetic_Biology_2006/IGEMideas%2005-29-06.ppt]
Protocols
To Do List
Since I have severe separation anxiety, post what you did and how things turned out each day right here. Then I can offer suggestions and help plan the next steps.
Monday
Plasmid Prep
Vector Digestions
Develop Assembly Schedule
Tuesday
Trevor: We will have to make reversed RBS and reversed double terminator - design PCR primers for this the way we did for the reversed pAra on Friday and post the proposed sequences here.
Making Reverse pARA
cloning hix fragments
Below are some questions from Jeff and some responses by Todd:
1. Can we double up on some constructions like Promoter(Forward) and Promoter(Reverse) by inserting them into a plasmid that already has a Hix site and getting a 50-50 split (which we can presumably separate out)?
I think it would be better to keep the two promoter orientations as separate constructions.
2. Could we start with a plasmid with a Hix site and insert several things at the same time and then sort them out later? (Example: could we put in a short promoter, a medium length RBS, and a long coding region at the same time and sort them out by length as to which connected? I think this is what you referred to as shotgun ligation when we were discussing the Traveling Salesman Problem.)
Again, I think that keeping things separate will be easier in the long run.
3. Should Hix be the LAST thing in any subconstruction so that we can create a version with HixL, one with HixR, and one with Hix C? I guess this boils down to the question of how big of a library of parts do we want to create. Is it the case that every time we want to have a Hix site, we need one of each type of Hix? If we take this approach, then we can investigate any differences between the types of Hix sites, perhaps finding the most effective/efficient combination.
The way I see it, there will be four pancakes, each in two orientations, and each one will have a hixC on the right (3') end. That way, each could be used in modular fashion to make any of the four pancake constructs we want.
I think that for now we should head for the construction of parts that can be used in the two different one-pancake devices that will work in the four-pancake ones. I think we should concentrate for now on using hixC only. The problem can be subdivided into the following three devices: Hin recombinase producer, pancake flipping operon, and recombination enhancer.
