Part:BBa_K1412614:Experience

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Protocol

Verification

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Activiation

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1. 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.

2. 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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1. Firstly, draw three dots on a plate before using oily pen from the bottom of culture dish.

2. Secondly, stab 3μl bacterium medium into the M63 semisolid medium at the dots.

3. Finally, culture the bacteria in constant temperature and humidity incubator at 37℃ and observe the condition of bacterium growth.

Measurement

The equipment we need is just a ruler!!!

Use a ruler to measure the diameter of colony from the bottom of the semi-solid medium. 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, R7…and record time and diameter. The migration radius caused

by chemotaxis grows higher over time, larger radius bringing less error. Each radius minus R1 is the net migration. Finally processing data

with excel.

Results

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Actually, we processing data 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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