McGill/Parts
From 2007.igem.org
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1. pLAC-rbs-luxI-STPO-pConstitutive(J23119)-rbs-luxR | 1. pLAC-rbs-luxI-STPO-pConstitutive(J23119)-rbs-luxR | ||
| - | + | *The following three parts were ligated together to create a construct R0062+B0034+C0060. A functional form of the aiiA gene for our system. This assembled part will be submitted to the registry.* | |
| - | + | 2. B0034 - 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) | |
| - | + | 3. C0060 - The aiiA gene. Would inactivate the autoinducer giving us more controle over our system. It is the heart of our construct. | |
| - | + | 4. R0062 - luxR promoter. The promoter we will be using to turn on expression of AiiA. | |
| - | + | 5. J40001 - one of the components of the not yet oscillating, oscillator. This component was consructed 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. | |
| - | 7. E0434 - 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. | + | 6. I15004 - 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. |
| + | |||
| + | 7. I5610 - this is the elowitx 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 constiuent parts which we confirmed from multiple gel runs. | ||
| + | |||
| + | 8. 15611 - a constiuent part of our new repressilaot 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! | ||
| + | |||
| + | 9. E0434 - 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. | ||
Revision as of 04:49, 25 October 2007
1. pLAC-rbs-luxI-STPO-pConstitutive(J23119)-rbs-luxR
- The following three parts were ligated together to create a construct R0062+B0034+C0060. A functional form of the aiiA gene for our system. This assembled part will be submitted to the registry.*
2. B0034 - 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)
3. C0060 - The aiiA gene. Would inactivate the autoinducer giving us more controle over our system. It is the heart of our construct.
4. R0062 - luxR promoter. The promoter we will be using to turn on expression of AiiA.
5. J40001 - one of the components of the not yet oscillating, oscillator. This component was consructed 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.
6. I15004 - 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.
7. I5610 - this is the elowitx 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 constiuent parts which we confirmed from multiple gel runs.
8. 15611 - a constiuent part of our new repressilaot 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!
9. E0434 - 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.
