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Team:XMU-China/Circuit
From 2013.igem.org
Overview
So we designed three plasmids to function as the three important parts in the oscillation circuit, and all of them are in the charge of the same quorum sensing promoter to make sure that all target genes share the same oscillatory period (Table 2-1).
| No. | Plasmid | ReplicationOrigin | CopyNumber | Resistance | Size(bp) | |
|---|---|---|---|---|---|---|
| Insert | Backbone | |||||
| A1 | pSB1C3-gfp-luxI | pSB1C3 | high(100~300) | Chloromycetin(Cm) | 4052 | 2070) |
| A2 | pSB1C3-sfgfp-luxI | pSB1C3 | high(100~300) | Chloromycetin(Cm) | 4046 | 2070) |
| A3 | p3H-GFP-luxI | p3H | Middle(18~22) | Ampicillin (Amp) | 4052 | / |
| A4 | p3H-sfGFP-luxI | p3H | Middle(18~22) | Ampicillin (Amp) | 4046 | / |
| B | pSB3T5-aiiA | p15A | Middle(10~12) | Tetracycline (Tet) | 2135 | 2837 |
| C | pSB4K5-ndh | pSC101 | Low(5) | Kanamycin (Kan) | 2658 | 3004 |
In a synchronized oscillation circuit, each A, B and C plasmid is necessary. The plasmid A expresses GFP reporter and LuxI proteins, which is the positive feedback in oscillator; the plasmid B expresses protein aiiA to degrade AHL and acts as the negative feedback in oscillator; the plasmid C expresses NAD-2 to generate H2O2 to communicate between colonies, is the coupling part in circuit (Fig 2-5).
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| Fig. 2-5 Plasmids A, B and C in oscillation circuit |
You may have noticed that these three plasmids have different resistant genes and replication origins on their backbone. Different resistant genes are used mainly for selection. And since different replication origin means a different copy number of target genes in this plasmid. Using different replication origins is to produce different target proteins in an appropriate ratio, and only in this way can oscillation be observed. Just like a glee needs Soprano, Tenor and Bass to cooperate to finish a song. Besides, three artificial plasmids with same copy numbers always have a competitive relation in one cell and cannot be well expressed.
Plasmid A has two different backbones with high and middle copy numbers respectively. We built them to see the copy numbers' effect on oscillation. Results can be seen in Exploration (For a better Glee).
All of the plasmids we constructed are confirmed by agarose gel electrophoresis, and result can be seen in Parts, where you can also get the link to parts we submitted.
After the onerous construction work, our glee is ready to perform the synchronized oscillation song, so we have to find them a stage. Let's go to see the beautiful opera in Microfluidics (Stages).
Reference
1. Prindle, A., et al., A sensing array of radically coupled genetic 'biopixels'. Nature 481, 39-44, 2012;
2. Pedelacq, J. et al., Engineering and characterization of a superfolder green fluorescent protein. Nature 24, 79-88, 2006;
3. Waters C. M. & Bassler B. L. Quorum Sensing: Cell-to-Cell Communication in Bacteria. Annu. Rev. Cell Dev. Biol 21, 319-46, 2005.
