Team:Chiba/Experiments:copy number

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Balancing Player

Design

Fig. 1 Player Balancing
Fig. 1 Player Balancing


In this section, we intended to create time-delay by altering the amount of luxR protein per cell.(*1) Since the amount of LuxR that receives AHL differs, we hypothesized that the time required to reach the threshold AHL concentration will differ.

To do this, we used different copy number plasmids of the Receivers. (*1)

By altering the copy number of plasmids in the receiver cells, the time required for the response is altered.
If the copy number is altered, the amount of LuxR produced by the receivers will also change.
The amount of LuxR that receives AHL will change, so the time required for AHL to reach a threshold level will be altered.



Experiment

We used a pMB1 ori high copy vector containing BBa_T9002 and a low copy vector containing BBa_T9002 with a P15A as ori.

We determined the effects of altering the plasmid copy number by comparing the above situations.

Experiments used the following genetic circuits:

  • Sender
  • Receivers
**BBa_S03623 (AHL autoinucer)

Image:LuxI-sender Chiba.gif

Image:High-Copy-Receiver Chiba.gif

  • Low Copy Receiver

Image:Low-Copy-Receiver Chiba.gif



In addition, a Chloramphenicol resistance marker-containing empty plamid was transformed to the strain containing BBa_T9002, and a Ampicillin resistance marker-containing empty vector was transformed into the low copy variant, both resulting in a double transformation.


Method

  1. Transformed sender (Ptet-LuxI), high copy receiver (Ptet-LuxR-Plux-GFP-pMB1) and Medium copy receiver (Ptet-LuxR-Plux-GFP-p15A) respectively into E coli strains (JW1908).
  2. Inoculated them independently in liquid media. Incubated at 37°C 12h.
  3. Inoculated again in Fresh liquid media upto about OD600=2 at 37°C
  4. Washed sender and receivers.
  5. Mixed them. (Sender:Receiver=1000μL:1000μL)
  6. Incubated at 30°C.
  7. Measured intensity of green fluorescence at regular time intervals. (Fluoroskan AscentR FL&Fluoroskan AscentR Thermo Electron Corporation)

Result & Discussion

Fig. 2 Time Delay Test: Highcopy Receiver & Reporter vs.Lowcopy Receiver & Reporter
Fig. 2 Time Delay Test: Highcopy Receiver & Reporter vs.Lowcopy Receiver & Reporter



Compared to GFP expression from (Ptet-LuxR-plux-GFP-pMB1), expression from (Ptet-LuxR-pLux-GFP-p15A) was greatly reduced (Fig. 2).

Possible explanations for this are:

  1. GFP expression was reduced since it was placed on a low copy number plasmid.
  2. The amount of LuxR synthesized was too small.

However, (Ptet-luxR-p15A + plux-GFP-pMB1) and (Ptet-LuxR-pLux-GFP-pMB1) resulted in exactly the same trasnfer Curve (Fig. 3).

Thus, we can deduce that the expression levels of LuxR from the p15A vector is sufficient to activate pLux from the high copy number vector. Explanation 1 is therefore more probable.

Fig. 3 Time Delay Test: Lowcopy Receiver & Highcopy Reporter vs.Highcopy Receiver-Reporter
Fig. 3 Time Delay Test: Lowcopy Receiver & Highcopy Reporter vs.Highcopy Receiver-Reporter




Demo ~Receivers~

Breif explanation on Methods:
1. LB pre-cultured Sender (BBa_S03623 (JW1908)) was mixed with LB-agar to produce sender containing bacterial plate.
2. Receiver colony was transfered to a nitrocellulose filter and placed on a Sender (BBa_S03623 (JW1908)) containing bacterial plate.
3. AHL produced by sender is diffused in the LB-agar plate and the receivers on the nitrocellulose filter synthesize GFP when they are stimulated by a certain amount of AHL.
4. Plates cultured at 37°C were exposed to UV (312nm) light once every 30 minutes to observe GFP fluorescence.

The receivers are:

  • AiiA synthesizing receiver.
  • receiver which has different copy number.
  • Cross-talk receiver.


Results

--->more about Receivers Demo experiments detail



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