The following are the biobricks that we have used for our project as well as new biobricks that we have synthesized, to be added to the iGEM Registry of standardized parts.
This is the modified repressilator which containing the same components as the I5610 repressilator with the addition of the degradation tag from E0434.
pLAC-rbs-luxI-STPO-pConstitutive(J23119)-rbs-luxR - This is the newly synthesized I15004 that has not yet been received. We added a RFP reporter so we can assay the oscillations using both the bricks in the system; and, inaddition, a constitutive promoter was added on the LuxR such that there would be less competion with the repressilator once incorporated into the system.
- one of the components of the not yet oscillating, oscillator. This component was constructed by members of the McGill team last year and has proved successful and robust in its developing capabilities. It contains a LuxR promoter controlling the expression of LacI. Additionally, this has a reporter CFP which is used to assay the output of the oscillating system.
- a constituent part of our new repressilator which contains all of the parts as above though lacking the LVA. for the YFP output. Alas, what can we do? We can put an LVA on!
- the second part of the not yet oscillating oscillator. Using this part directly from MIT reared some problems as the proper bands were not attained and when coupled with the J40001, the predicted results were again, not attained (see lab notebook portion). This brick, from MIT, contains the LacI generator promoter regulating LuxI protein along with a cI LVA. Additionally, there is a LuxR protein, the constituent of the AI, under the control of a tetracycline promoter. Due to the lack of sucess from this brick, we synthesized a new gene with the same basic components though with a few extras: a constitutive promoter for the Lux R, an RFP, and a lot lesser spacer DNA.
- We made this construct with LuxR/HSL promoter R0062, B0034 RBS, and the C0060 aiiA gene in order to give us more control with our repressilator system. The aiiA gene will be turned on by the LuxR/HSL promoter in the presence of the LuxR activator protein complexed with the autoinducer 3-oxo-hexanoyl-HSL.
- The I724000 brick combined with the J40001 brick. This brick will be created to facilitate the transformation of our oscillator parts into the same cells.
- the source of the LVA which is a simple YFP protein with the LVA thus, we simiply cut out the LVA from this part and pasted it into the I5611... or not so simply.
- The aiiA gene. Would inactivate the autoinducer giving us more control over our system. It is the heart of our construct.
- The rbs for our Aiia construct. Small and difficult to work with (especially when you don't realize that it isn't already included in the aiiA gene itself)
- luxR promoter. The promoter we will be using to turn on expression of AiiA.
- this is the Elowitz represillator which is ultimately to be coupled to our oscillating system. This system contains three parts: cI lambda protein regulator through a tetracycline promoter, a lacI protein regulated under a cI lamda promoter, and a tetracycline protein regulated under a lacI promoter. Obviously, this is a triple repressing system leading to robust sinusodial curves when coupled to the oscillator. As we have seen before, the 15610 from MIT was not as they said it was. Through multiple gels and experimentation, it was concluded as such (see the lab protocol). So, we decided to make a new repressillator from is constituent parts which we confirmed from multiple gel runs.