Difference between revisions of "Part:BBa K1412614:Experience"

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Protocol

Verification

Activiation

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

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℃.

Measurement

What 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

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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