Part:BBa_K1412614:Experience

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Protocol

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Genetic link stage

1. As a skeleton, TT terminator link with target gene CheZ.

2. Then link the skeleton to target gene CheZ-TT.

3. Next, link RBS-CheZ-TT with promoter Plac.

Verification

Characterization stage

1. Transfer pBAD-RBS-CheZ-TT (BBa_K1412624) into competent cell of E.coli (ΔCheZ) respectively.

2. Coated plates(LB solid medium with the antibiotic concentration 50ug/ml chloromycetin) and culture in 37℃ biochemical incubator for 12h.

3. Select colony to culture in 5ml LB fluid medium, of which the chloromycetin, antibiotic concentration is 50ug/ml.

4. Activation of bacterium: transfer 50uL bacterium medium into a new 5ml LB fluid medium, of which the chloromycetin, antibiotic concentration is 50ug/ml.

5. Stab of bacterium: We draw three spots on a plate before, then stab 3ul bacterium medium into the M63 semisolid medium at the spots.

6. Culture the bacteria in constant temperature and humidity incubator at 37℃.

Measurement

1. The initial colony radius is recorded as R1, and the radius are measured at 12h, 24h, 30h, 36h, 42h... as R2, R3, R4, R5, R6, … The migration radius caused by chemotaxis grows higher over time, larger radius bringing less error. Each radius minus R1 is the net migration radius at certain time. Plot the net chemotaxis radius versus time and set the radius of pLac-RBS (1.0)-CheZ-TT (BBa_K1412000) as RBS strength 1.0. Then we can calculate the RBS efficiency (ΔR2L/ΔR2M) and (R2J/ΔR2M) and compare them with the official data.

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