Team:IIT Delhi/Circuit Design
Circuit Design and Construction
Two circuits were central to our work done in the project. The first one was the 5n1, which was a 5 node oscillator constructed by Richard Murray et al, that was not modified to produce square waves. The 5n1 (560) was a gift from Richard Murray (Addgene plasmid # 70730), and had the following topology and circuit –
Further, apart from this system, that was ordered for testing our characterization and measurement devices, we designed our own novel 5 node ring oscillator, which contained modifications that are mentioned at the end of the section. The topology and circuit that we designed and constructed are as follows –
This circuit was constructed in part through cloning, and in part through synthesis via IDT and Genscript. Further, the salient features of this circuit are –
- Cooperativity of repressors – Cooperativity was central to our work done, in ensuring that the rise and fall is fast for our circuit. The cooperativity of the repressors in the system that we have constructed is –
NODE
REPRESSOR USED
COOPERATIVITY REPORTED
A
Orf2
6.1
B
TetR
3
C
SrpR
3.2
D
PhlF
4.5
E
BM3R1
4.5
- None of the genes contain degradation tags, since they are a source of noise. The dynamics still remained square despite this change, which was modelled by us and verified via bifurcation analysis coupled with a squareness coefficient to quantify each point in the parameter space. For more details, refer to the modeling section. However, the shape of the waves that we obtained for a specific set of parameter values (even after removing degradation, i.e.- reducing the negative term in the differential equations in the model) are –
- The reporter (GFP) has been placed on a low copy number plasmid (the same plasmid as that of the rest of the circuit). The plasmid used was pSB3T5, which contains the p15A origin of replication, giving a copy number of 5-15.
Further, apart from this system, that was ordered for testing our characterization and measurement devices, we designed our own novel 5 node ring oscillator, which contained modifications that are mentioned at the end of the section. The topology and circuit that we designed and constructed are as follows –
This circuit was constructed in part through cloning, and in part through synthesis via IDT and Genscript. Further, the salient features of this circuit are –
- Cooperativity of repressors – Cooperativity was central to our work done, in ensuring that the rise and fall is fast for our circuit. The cooperativity of the repressors in the system that we have constructed is –
- None of the genes contain degradation tags, since they are a source of noise. The dynamics still remained square despite this change, which was modelled by us and verified via bifurcation analysis coupled with a squareness coefficient to quantify each point in the parameter space. For more details, refer to the modeling section. However, the shape of the waves that we obtained for a specific set of parameter values (even after removing degradation, i.e.- reducing the negative term in the differential equations in the model) are –
- The reporter (GFP) has been placed on a low copy number plasmid (the same plasmid as that of the rest of the circuit). The plasmid used was pSB3T5, which contains the p15A origin of replication, giving a copy number of 5-15.
| NODE | REPRESSOR USED | COOPERATIVITY REPORTED |
| A | Orf2 | 6.1 |
| B | TetR | 3 |
| C | SrpR | 3.2 |
| D | PhlF | 4.5 |
| E | BM3R1 | 4.5 |
