Part:BBa_K1412614:Experience

Protocol

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.

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.

5. Observe the the condition of bacterium growth,and prepare a ruler.

6. 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.

7. 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….

<b>Figure 1. Figure 4.

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.

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