Difference between revisions of "Part:BBa I718008:Experience"

Revision as of 22:57, 4 October 2011

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_I718008

This Cre recombinase generator can be used to induce recombination between two lox site at a tunable rate. An experience conducted by iGEM Paris 2007 gives the recombination rate without induction:

A strain bearing the cassette lox-kanR-lox inserted into its chromosome was transformed with BBa_I718008 in the pSB1A2 plasmid. Directly after transformation, liquid cultures were launched with either Amp or Amp+Kan. 100µl of the cultures were regularly plated at appropriated concentrations on LB+Amp, giving the following growth curves. The recombinated cells loose their resistance to Kanamicyn and die making the growth slower.

In July 2010, the Davidson iGEM team used this part from both the 2010 and 2009 kit plates distributed by MIT and found that they were not the right part. Our goal was to ligate pBad-RBS-Cre as a vector to pTet-loxP-RBS-RFP-loxP insert and other variations of lox sites to determine what mutated versions of lox would recombine in the presence of Cre. However, we were alarmed when our negative controls turned red several times and with several people attempting this transformation (since negative control only contained pBad-RBS-Cre). We also kept seeing a 1kb band with both colony PCR screens and digestion with EcoRI and PstI. We investigated further by digesting with PvuI and PstI since Cre has an internal PvuI site along with pSB1A2. We expected to see at least two bands but found only one. The registry did not send out this part where the depository says it is located.

In September 2011, Tokyo_Tech iGEM team used this part from 2011 kit plates distributed. It was used for meditating recombination at lox2272 cassette of BBa_K649201 and lox71/66 cassette of BBa_K649202. We verified the sequence of this to be right before use. In in vivo assay of K649201 and K649202, recombination took place that the part was proven to work. However, the promoter of this had a serious problem to be used as an inducible promoter. Because of the severe leaking of PBAD/araC, the cre expression level of uninduced sample was almost as same as induced. To get more information about the experiment, please see BBa_K649201[[1]] or BBa_K649202[[2]] registery page.

Cre-meditated recombination at lox2272 cassette. Period of 0.5hr. The leftmost is a negative control which don't have Cre-expressing plasmid. The center is an arabinose induced sample which has both Cre plasmid and BioBrick BBa_K649201. The rightmost is a uninduced strain which has both plasmid like as the center.

Cre-meditated recombination at lox71/66 cassette.Period of 0.5hr. The leftmost is a negative control which don't have Cre-expressing plasmid. The center is an arabinose induced sample which has both Cre plasmid and BioBrick BBa_K649202. The rightmost is a uninduced strain which has both plasmid like as the center.

User Reviews

UNIQ61777903343aff3e-partinfo-00000000-QINU UNIQ61777903343aff3e-partinfo-00000001-QINU