Oscillator Example

From 2006.igem.org

Home
Team 2

Here is an example of a 3-way polycistronic oscillator that should oscillate between cyan (ECFP), yellow (EYFP), and red (RFP)

the promoters are Lac, Tet, and pBad/araC, controlled respectively by the proteins LacI, Tet (+tetracycline), and araC

One-third of our work is done for us: we can use the composite part Bba_I14029 (check status: planning?)

its structure is lac promoter--strong rbs--TetRGene--strong rbs--ECFP the part numbers: R0010 B0030 C0040 B0030 E0022

Let's assume for now that we want to use strong RBSs for all our constructs. Then we'll want to make

                 tet promoter--rbs--AraCgene--rbs--EYFP
                   R0040       (B0030 C0080) (B0034--EYFP--STOP) is available as E0430   
                               (composite part I14010, status is planning)

the EYFP is non-destabilized because the ones with degradation tags all seem to have issues! we have been warned!

Now our work is to amplify the bricks R0040, I14010, and E0430 and stick them together

I couldn't find a two-stage procedure. anyone have better luck??

and pBad/araC promoter--rbs--LacI--rbs--RFP--STOP)

                 (I0500)             (I14022 ) (J06703         )

again it has 3 parts. There seem to be issues with both of the last two: I14022 is "planning," and J06703 is "missing." The latter is a clone of something that was "returned with sequence resembling nothing like the desired sequence." Oh really??