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Part:BBa_K1412614:Experience

Designed by: Ruihua Zhang   Group: iGEM14_XMU-China   (2014-10-01)


Protocol

Verification

Figure 1 Verification of plasmid BBa_K1412614 and BBa_K1412014.

Activiation

1. Transfer 50 μL bacterium solution (pLac-RBS (1.0)-cheZ-TT, pTet-RBS (1.0)-cheZ-TT, pBAD-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 & Measurement

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.

Figure 2. The schematic diagram of how we measure the diameter of colonies.

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

Figure 2A.Culturing with 0.02 L-arabione for 48 hours, distinguish difference of chemotaxis diameters between each colonies is shown. Figure 2B. The relative activity of different promoters to pLac.


Firstly, 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). As we can see, 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 could tell us about the relative promoter activity of pBAD (BBa_K206000) to pLac. While L-arabinose can induce pBAD, We characterize the relative activity of pBAD 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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