Chiba/Making Marimo

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Revision as of 04:32, 27 October 2007

Making Marimos

Fig1. Scheme of final gene cuircuit for Bacteria Marimo.

Parts Construction

Because we found that FliC code includes restriction enzyme(EcoRI,SpeI,PstI) which is used in Biobrick, we divided into plasmid of FliC and one of signal, and aimed the double transformation.

Moving FliC-His generator

Experiment

Fig2. Moving FliC-generators into p15A plasmid (not biobrick!)

• We regulate the expression of His-tagged FliC by lux promoter. Namely, if LuxR is expressed, bacteria can express FliC via Quorum Seinsing.
  
• Because the Quorum Sensing device is on ColE1-type vector, we need to export the FliC unit into the plasmids with compatible origins such as p15A.

Results

First attempt to import FliC-His generator into p15A vector (see Fig).

• Communication units are on ColE1 type plasmids. To make this stickly FliC construct compatible with communication circuits, this is an absolute necessity.....
  
• Not yet (as of 10/26/2007). The first ligation sucked. Cloning is still underway.
  
• Many forbidden sites for Biobrick production throughout the FliC gene. We are planning to eliminate them all in near future by repeated site-directed mutagenesis using ExSite method.

FliC-his biobrick

Experiment

• Making Biobrick version of Flic-His and other stickly-FliCs. unfortunately, FliC has many EcoRI,SpeI,PstI sites. We have to eliminate them one by one by site-directed mutagenesis.