Difference between revisions of "Part:BBa K1412614:Experience"
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| − | Set the diameter of the colony with promoter Lac as 1.0, and plot the data in excel, We got the following table (Figure 3). | + | Set the diameter of the colony with promoter Lac as 1.0, and plot the data in excel, 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. | ||
| − | |||
| − | |||
[[File:Figure_3._The_relative_activity_of_different_promoters-1.jpg |500px]] | [[File:Figure_3._The_relative_activity_of_different_promoters-1.jpg |500px]] | ||
Revision as of 06:36, 17 October 2014
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how you used this part and how it worked out.
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
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
1. Observe the the condition of bacterium growth,and prepare a ruler.
2. When we can see a visible difference of the diameter of semi-solid medium with time pass by, begin to measure the diameter of bacterium
colonies from the bottom of the semi-solid medium to avoiding the collapse of semi-solid medium.
3. Recording data: The diameter of initial colonies(0h) is recorded as R1. And the diameter of the colonies at 12h, 24h, 30h, 36h, 42h... as
R2, R3, R4, R5, R6, R7….
Results
Set the diameter of the colony with promoter Lac as 1.0, and plot the data in excel, 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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