Difference between revisions of "Part:BBa K1412614:Experience"
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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 | + | 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 |
| − | whose chloromycetin concentration is 50ug/ml to culture for 12 hours in 37℃ horizontal rotators at 200 rpm. | + | 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'''=== | ==='''Culture'''=== | ||
Revision as of 05:22, 17 October 2014
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Protocol
Verification
Activiation
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
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
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
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.
Applications of BBa_K1412614
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