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Team:Princeton/ProjectDetails
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===Surface Patterning=== | ===Surface Patterning=== | ||
Revision as of 07:21, 29 October 2008
PRINCETON IGEM 2008
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Contents |
Plasmid Designs
We worked on designing and constructing several plasmids this summer. Our plasmids were on the whole designed using PCR SOEing (Splicing by Overlap Extension). The process involves PCRing the pieces of the plasmid insert separately, but adding one or more "overlap" regions to each insert section that consists of the end sequence of the neighboring section. For example, if the insert to be put into the vector is A-B-C, then A would be PCRed with a bit of the beginning of B added onto the end using a primer overhang. B would be PCRed with a bit of the beginning of C added on (the end of A need not be added onto the beginning of B because B already has an overlap with the beginning of its sequence that was added onto the end of A). Then A could be "SOEed" to B by running a PCR reaction of equimolar amounts of A and B, the forward primer of A and the reverse primer of B. A and B will anneal to each other and acts as primers in addition to the oligos in the reaction. B and C could similarly be SOEed, followed by A-B and B-C. A and B and C could also be SOEed together in a single reaction, with every piece acting as primer, but there are decreasing outputs the more pieces that one attempts to SOE in a single reaction.
Below is the list of plasmids we have designed and worked on, each linking to a design page. Some plasmids were redesigned, and hence appear more than once, with a number tacked on the end. At the top of each page the name of the plasmid is listed, followed by the vector, with the position and names of the restriction sites used to cut, and the insert, similarly marked with restriction sites. If the enzymes do not match it they have compatible overhangs.
The highlight code is as follows:
| yellow: overlap of primers with the DNA | dark green: restriction site | pink: "junk" sequence to allow restriction enzymes to cut near the end of a strand. | light blue: kozak sequence. | dark blue: stop codon. | gray: a 2A sequence (the amino acid sequence is cut by the cell) | red: either a linker to fuse two proteins (where indicated) or a sequence with a restriction site removed by changing a base pair with the primer. |
List of Plasmid Designs
pLV-Hef1aLacO-MashI-2A-ChAT-Ubc-Hyg
pLV-Hef1aLacO-MashI-2A-Nurr1-Ubc-Hyg
pLV-Hef1a-LacO-MashI-Cerulean-Ubc-Hyg
pLV-Hef1a-LacO-MashI-EGFP-Ubc-Hyg
pLV-TRE-GLRA1-2A-gephyrin-Ubc-Neo
pLV-TRE-GLYT2-2A-Viaat-Ubc-Bla
pLV-TRE-Lbx1-2A-GLRA1-2A-D5R-Ubc-Bla
pLV-Ubc-rtTA-2A-LacIKrab-2A-LuxR-IRES-Puro
pLV-Ubc-rtTA-2A-LacIKrab-IRES-Puro
PCR SOEing and Sequencing Primers
We created over 100 primers for the construction and sequencing of our plasmids. For the sequences and properties of each primer, as well as which primers were used for which plasmids, click here.
Experiments
See Experiments section.
Surface Patterning
It has been shown that plating neurons in a specific pattern gives the emerging networks special properties, which are determined by those patterns. Also, the growth of these networks is strongly influenced by the patterns they are allowed to grow in. Therefore, by patterning the surfaces that neurons grow on, we can direct specific network connections. This is at the heart of the architecture of the toggle switch.
