Part:BBa_K1412614:Experience

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Protocol

Verification

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Activiation

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Transfer 50 μL bacterium solution (pLac-RBS (1.0)-CheZ-TT, pLac-RBS (0.01)-CheZ-TT, pLac-RBS (0.3)-CheZ-TT) into 5 ml new LB liquid medium

whose chloromycetin concentration is 50ug/ml to culture for 12 hours in 37℃ horizontal rotators at 200 rpm.Then transfer another 50μL

bacterium solution from the LB above to another new 5ml LB liquid medium whose chloromycetin concentration is 50ug/ml to culture in 37℃

horizontal rotators at 200rpm for 3 hours.

Culture

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We draw three dots on a plate before, then stab 3μl bacterium medium into the M63 semisolid medium at the dots.

After that, culture the bacteria in constant temperature and humidity incubator at 37℃.

Results

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Actually, we kept measuring chemotactic diameters of three colonies at different time and set the diameter of the colony with promoter Lac as

1.0. We got the following table (Figure 3). The ratio between each colony diameters was fixed after 36 hours. If we set the fixed ratio as

relative promoter activities, from our characterization, promoter TetR (BBa_R0040) activity is 1.86 relative to promoter Lac (BBa_R0010).

Refer to published papers [1] [2], promoter activity between pTetR and pLac has already been measured, and their ratio (pTetR/pLac) is 1.58.

So our system is reliable as it could tell the difference between different promoter activities. However, no published data tell us about the

relative promoter activity of pBAD (BBa_K206000), while L-arabinose can induce pBAD (BBa_K206000). The characterization of the relative

activity of pBAD is carried out with 0.02% inducer L-arabinose in culture. And the ratio (pBAD/pLac) is 0.37.

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